For the M2 Macrophage Generation Medium; it is extremely important that the shelf life of 2 weeks (after addition of the cytokines) is not exceeded; the yield will quickly decrease thereafter. It is best to use the M2 medium as fresh as possible to avoid discrepancies between M1 and M2 yield.

Spinning the cells for 15 min at 350 x g has been proven and tested by PromoCell Research & Development. The QC department uses these settings during testing. Any lower centrifugation value (g-force and/or time) will lead to significant cell loss by means of non-sedimented; but intact; macrophages.

Yes; our Lymphocyte Separation Medium 1077 is endotoxin-tested. The specification for the product is < 10EU/ml.

Yes we have received a customer feedback that our 3D Tumorsphere Medium XF (C-28070) has been successfully used for tumorsphere formation of mouse cell lines.

MSC Growth Medium 2 (C-28009) is an optimized medium formulation with reduced serum content to allow for more standardized culture conditions (considerably lower lot to lot variation). You can replace MSC Growth Medium by MSC Growth Medium 2. Coating of culture vessels is not necessary. We recommend to plate the cells (hMSCs from bone marrow; adipose tissue; or umbilical cord) at 4;000 cells/cm².

a) We usually perform macrophage differentiation in T75 flasks and 6-well plates. We haven't tested differentiation in smaller formats. But we assume it will be problematic to thoroughly wash the surface of the wells to remove non-adherent cells after the attachment phase. b) Detachment of the mature macrophages is possible but re-attachment can lead to significant cell loss (30-50%). Please also keep in mind that working in 96/384 well-format has some inherent drawbacks (e.g.; evaporation of media; dry wells; etc.).

This method produces large numbers of adherent monocytes in only 1.5 h. If the washing steps are properly performed 80-90% purity can be expected. The attached cells are “untouched” since no binding of magnetic microbeads has occured. This also excludes phagocytosis of the microbeads by the monocytes an event which is unfavorable with regard to cellular health. In addition the adherence method is time-saving and cost-effective.

There is no significant difference in the percentage of cells that will differentiate into Dendritic Cells. But when using cryopreserved cells the initial cell loss will be higher compared to when fresh cells are used. i.e. the final number of differentiated cells that can be expected will be higher with fresh cells as a starting material due to lower cell death rate.

You should use complete DC Generation Medium/DC Generation Medium XF (with all the cytokines). As cells are metabolically active media should be changed every 3 days. We have observed that the dendritic cell phenotype remains stable for up to 7 days.

The Macrophage Detachment Solution (C-41330) directly affects the cell membrane. HSA in the Wash Buffer supports regeneration of the cell membrane and protects the cells during the critical phase directly after detachment from detrimental effects.

At PromoCell we guarantee for our primary human cells ≥ 500;000 viable cells after thawing. For this; we dispense > 500;000 cells per cryovial before cryopreservation as there will always be a certain percentage of dead cells after freeze/thaw. In order to know the number of cells that survived the procedure; we defrost a representative number of vials per lot during QC; determine the cell viability using an electronic counting device and then calculate the number of viable cells that can be recovered after thawing. Both numbers - the calculated number of viable cells and the viability - can be found on the lot-specific Certificate of Analysis (CoA) that can be downloaded from our website. Example: When the CoA indicates 600;000 viable cells and a viability of 80%; this means that the vial actually contains 750;000 cells (viable + dead); 80% thereof (600;000) were viable after thawing in our QC. We do not indicate the total number of cells per vial but just the number of expected viable cells which can be recovered when the recommended thawing protocol is used. You don't have to calculate any viabilities by yourself. When the recommended plating density for your cell type is 5;000 - 10;000 cells/cm²; then the 600;000 viable cells can be plated e.g. in a T75 (corresponding to 8;000 cells/cm²) or in a T75 + a T25 (corresponding to 6;000 cells/cm²).

Baculovirus is generally used in conjunction with insect cells (Sf-9; Sf-21) to produce recombinant proteins (cytokines; growth factors). None of our Specialized Media (Media for Primary Human Cells; Blood and Stem Cell Media; Cancer Cell Media) contain recombinant proteins produced in insect cells. This also applies to our Cryo-SFM Freezing Medium.

According to our experience; they can be maintained for 3 to a maximum of 7 days. However; the morphology will change and they will look more and more "degenerate".

The Vena saphena section that we use for HSaVEC isolation originates from the thigh.

All our Human Uterine Fibroblasts (HUF) are prepared from the myometrium.

Some of our customers have successfully used PromoCell Endothelial Cell Growth Medium MV2 (C-22022) when co-culturing Human Coronary Artery Endothelial Cells (HCAEC; C-12221) and Normal Human Dermal Fibroblasts (NHDF; C-12300).

Our HCASMC (C-12221) are isolated from the large arteries; i.e. from

Right coronary artery
Left main coronary artery
Circumflex coronary artery and
Left anterior descending coronary artery.

Yes primary mouse keratinocytes grow well in this media when you reduce the CaCl₂ concentration to 0.025 mM - 0.05 mM CaCl₂ (optimal Ca concentration for primary human keratinocytes is 0.06 mM).

FBS (or BSA) can be substituted for HSA; however we do not recommend this as the FBS will lead to unpreferable immunologic stimulation of human macrophages.

The addition of EDTA to the PBS is to further augment the “anti-clumping” activity of the Ca²⁺/Mg²⁺ free PBS.

Usually we do not recommend specific plasticware but we have found that with PromoCell’s Dendritic Cell and Macrophage Generation Media choice of plasticware can have a lot of an influence. For our Dendritic Cell Generation Media we recommend to use tissue culture vessels from BD Falcon^TM.

Usually; we do not recommend specific plasticware but we have found that with PromoCell’s Dendritic Cell and Macrophage Generation Media; choice of plasticware can have a lot of an influence. For our M1-/M2 Macrophage Generation Media XF we recommend the Nunc plasticware with Nunclon surface - as not only will the detachment efficiency vary (up to 20%); but also the efficiency of the differentiation process itself may be altered.

Samples in RNAlater will NOT freeze at -20°C they are liquid at 4°C and -20°C. The samples can be stored at 4°C for up to 1 month and are stable at room temperature for up to one week. At -20°C they can be stored indefinitely.

Our customers have successfully used TC flasks and dishes from all the leading cell culture plastic suppliers to grow PromoCell's primary human cells. We do not have any knowledge whether the dishes from local TC plastic suppliers work in the same way. We recommend to first test whether these brands provide the same good performance as the plastic of the leading manufacturers.

Both cytokines are produced in E.coli.

Yes, our MSC Growth Medium XF (# C-28019) contains phenol red. The concentration is confidential.

But we also supply a phenol red-free variant: Mesenchymal Stem Cell Growth Medium XF, prf (# C-28018).

The differentiation rate into the osteogenic lineage is 70-100%.

Problems in obtaining RNA with good yield and purity from mononuclear cells (hMNCs) are quite common. The reason for this is the large amount of free genomic DNA usually contained in MNC preparations. This DNA originates mostly from granulocytes which underwent lysis during the isolation of the MNC. The granulocytes are gone in the final MNC preparation; but their genomic DNA - released during cellular lysis - is still there "sticking" to the MNCs. Solution: Remove DNA prior to RNA purification by a DNase digestion step. Most commercial systems include the option for such a DNase digest.

Our MSC Growth Medium XF (Ready-to-use) provides a xeno-free culture system for human MSCs. It contains all growth factors and supplements except attachment- and spreading factors. Therefore; culture vessels must be precoated with 10 μg/ml human or bovine fibronectin. Protocol: Fibronectin coating Dilute the Fibronection Solution to 10 μg/ml final concentration in Dulbecco´s PBS w/o Calcium and Magnesium. Overlay the culture surface of your tissue culture vessel with an amount of the diluted Fibronectin Solution sufficient to effectively coat the complete surface. Be sure that the entire surface is covered. Place flasks on a level surface at RT for 60 min. Aspirate the excess Fibronectin Solution and use immediately or let air-dry the open vessel under a laminar flow bench. Unused vessels may be stored at 4°C for up to 2 weeks.

Usually 90-100% of the hMSC show a neuronal morphology after differentiation with our Mesenchymal Stem Cell Neurogenic Differentiation Medium (C-28015). 60-80% of them are positive for nissl bodies after a nissl stain. However the differentiation capacity depends on the origin of the cells and the number of population doublings they have undergone.

The PromoCell DC Generation Media have been developed for the easy and efficient generation of immature as well as fully mature myeloid dendritic cells from peripheral blood monocytes. For freshly isolated mononuclear cells (MNC) and monocytes we recommend our DC Generation Medium XF (C-28052); for cryopreserved monocytes our DC Generation Medium (C-28050). When using DC Generation Medium (C-28050) with fresh MNC; the Monocyte Attachment Medium (C-28051) is needed in a first step for efficient adherence of the monocyte fraction. Please see Instruction Manual; Application Note and the graph below for further details.

Our DC Generation Medium XF (C-28052) has a xeno-free formulation. It provides a complete media system (ready-to-use all cytokines included) and shows efficient and reproducible in vitro maturation of moDCs from freshly isolated peripheral blood monocytes.

It takes 7-8 days to generate fully mature myeloid dendritic cells.

The Monocyte Attachment Medium allows for the efficient adherence selection (within 1 hr) of monocytes from freshly isolated mononuclear cells while maintaining optimal cell health. The Time-consuming and costly immunomagnetic purification of monocytes prior to DC generation is not necessary and can be skipped when using this medium.

DC Base Medium (C-28053) consists of 250 ml Basal Medium + SupplementMix. It does not include cytokines and must therefore be supplemented according to the user's individual needs.

DC Generation Medium (C-28050) is ready-to-use and consists of 250 ml Basal Medium + SupplementMix + cytokines.

DC Base Medium XF (C-28054) consists of 250 ml Basal Medium + SupplementMix. It does not include cytokines and must therefore be supplemented according to the user's individual needs.

DC Generation Medium XF (C-28052) is ready-to-use and consists of 250 ml Basal Medium + SupplementMix + appropriate cytokines.

Both; Base Medium XF and Generation Medium XF have a xeno-free formulation.

The pH of our Endothelial Cell Basal Media MV/MV2 is 7.4 ± 0.1.

NHEK.f are isolated from tissue samples (foreskin) of single donors; aged between 1-10 years. NHEK.f pooled are prepared from the foreskins of 3 individual donors. The cells of each donor are expanded in separate TC vessels and the cells are pooled after secondary culture; before cryopreservation. Both NHEK from single donor and NHEK pooled are in P2 after thawing.

Differentiation of hMSC into mature adipocytes takes approx. 2 weeks. You can keep the adipocytes for up to 3 weeks in the MSC Adipogenic Differentiation Medium. After 3 weeks we recommend to switch to PromoCell's Adipocyte Nutrition Medium (C-27438).

Most of our subcutaneous preadipocyte lots achieve > 80-90% differentiation when differentiation is induced at P2 (directly after thawing). We generally recommend using cells for differentiation tests that haven't undergone more than 4-5 doublings (a maximum of 1 passage after thawing); as the differentiation ratio will decline with the age of the cells.

The source is porcine pancreas.

The source of our heparin is ex porcine mucosa.

Our Human Renal Cortical Epithelial Cells (C-12660) are isolated from the cortex of the human kidney. The renal cortex is the outer portion of the kidney. It contains the renal corpuscles the proximal and distal convoluted tubules and the cortical collecting ducts.

The cells sink down to the bottom of the culture vessel but don't really attach. They retain a roundish morphology and can be rinsed off with culture medium easily.

Our HREpC (C-12665) comprise a heterogenous mixture of renal epithelial cells isolated from cortex and medulla. The HRCEpC (C-12660) are derived from renal cortex only.

The population doubling times for PromoCell hMSC (hMSC-BM; hMSC-AT; hMSC-UC) are typically ≤ 30 hrs when using Mesenchymal Stem Cell Growth Medium 2 (C-28009). If you seed the hMSC at 4.000 cells/cm²; it will take between 4-7 days until they reach subconfluency.

PromoCell's Normal Human White Preadipocytes (HWP) are isolated from adult subcutaneous or visceral adipose tissue from different locations. The cells are frozen in our serum-free freezing medium (Cryo-SFM) at the end of passage 1 (= secondary culture). A randomly selected vial is then used for quality control; which includes determination of growth characteristics; control of morphology; and tests for differentiation capacity into mature adipocytes. The recommended seeding density of preadipocytes after thawing/trypsinization is 5;000 cells/cm²; cells should be trypsinized before reaching 90% confluence. Population doubling times are usually between 20-50 hrs (10 population doublings guaranteed). Using a 1:4 split ratio; you can perform ∼4-5 passages with the cells. Preadipocyte Growth Medium (C-27417) is used to propagate the cells. To induce differentiation of preadipocytes into adipocytes; cells are grown in PromoCell's Preadipocyte Growth Medium until they reach 100% confluency. Cells are then cultured in Preadipocyte Differentiation Medium (C-27437) for 72 h; followed by 10-14 days in Adipocyte Nutrition Medium (C-27439). During this time the cells start to accumulate fat droplets which can be visualized under the microscope. We recommend performing differentiation experiments at population doubling numbers lower than 4-5; in order to reach a high differentiation level of the culture.

No; we don't perform CD90 immunomagnetic separation with our pericytes. We can however exclude fibroblast contamination as follows: 1) The presence of fibroblasts in our pericyte cultures would be detectable shortly after cell isolation; as pericytes need up to 2 weeks before they start proliferating. Fibroblasts on the other hand would proliferate immediately and overgrow the culture. 2) Characterization of the isolated pericytes during QC includes flow cytometry of CD146. Pericytes express CD146 whereas placental fibroblasts don't.

Our hPC-PL (C-12980) are isolated from microvessels of the human placenta; from the chorionic villi. The number of populations doublings is not determined for each individual cell lot; but in our experience; they can be grown for at least 15 population doublings.

Most primary cells detach within 5-10 min at 37°C. Inactivation isn't required but we recommend to centrifuge the cell suspension to remove accutase and EDTA before replating the cells.

Yes the specialized PromoCell media already contain the optimal amount of L-glutamine. Please don't add extra L-glutamine as this can be toxic for the cells.

PromoCell HUVECs are freshly isolated from umbilical veins. They are cryopreserved at the end of primary culture. After revival; they can be propagated for at least 15 doublings and will senesce eventually. HUV-EC-C from ATCC is a hypodiploid human cell line of endothelial origin (umbilical vein). The modal chromosome number is 45 occurring in 72% of cells counted. The rate of polyploid cells is 15.8%. The cells have a life expectancy of 50 to 60 population doublings. This indicates that the cells are no longer "normal cells" but have undergone some degree of transformation.

Our HUVEC single donor (C-12200) are isolated from a single umbilical cord; propagated in primary culture; and frozen down at subconfluency. For the preparation of HUVEC-pooled (C12203); we simultaneously isolate the cells from 2-4 umbilical cords and grow them in separate tissue culture dishes. The cells are pooled after trypsinization given that their growth rates are comparable. After thawing; our HUVECs (single donor and pooled) are both in P1. The recommended media are Endothelial Cell Growth Medium (C-22010) or Endothelial Cell Growth Medium 2 (C-22011). With respect to cell growth; HUVEC-pooled tend to have a more heterogeneous morphology with slightly more elongated cells but the doubling times are comparabel for both types (typically 18-36 hrs per doubling).

If you are growing our NHEK in PromoCell Keratinocyte Growth Medium 2 (C-20011) or Growth Medium 3 (C-20021) you don't need any feeder cells. The cells will grow as a monolayer in conventional tissue culture flasks.

Supplement Mix for Melanocyte Growth Medium M2 (C-39420) is free of serum and doesn't contain any PMA. The qualitiative and quantitative composition is proprietary.

Note: Melanocyte Growth Medium M2 and NHEM M2 cells were disontinued at the end of 2024.

Our HDMEC are isolated from the dermis of juvenile foreskin or adult skin. The purity is > 95%. Since the dermis contains blood and lymphatic capillaries; HDMEC cultures comprise blood and lymphatic microvascular endothelial cells that have differing morphologies. Both cell types have a common origin and can be identified by several markers. The ratio of lymphatic and blood derived endothelial cells can vary from lot to lot and is not determined at PromoCell.

The time needed to detach our primary cells depends on many different factors like the cell type; cell density; lot #; trypsin concentration; the efficiency of the washing step before adding the trypsin and the trypsinization temperature. For most cell types we recommend trypsinization at room temperature and direct observation of detachment under the microscope. This way; you can find out your individual trypsinization time and keep the contact time between cells and trypsin to a minimum. Most cells detach after 2-8 min. Please refer to the instructions in the Manual. For some cell types; trypsinization at 37°C or the use of Accutase or another Detachment Solution is recommended.

PromoCell Blood and Blood Progenitor Cells are cryopreserved directly after isolation (= P0). They haven't been in culture before freezing.

We use a classification system similar but not identical to the Fitzpatrick Skin Classification. The Fitzpatrick classification has six different categories (phototypes I-VI) which correlate with the level of skin pigmentation (melanin) and sunburn following sun exposure. Fitzpatrick I corresponds with the lightest of skin complexions, while Fitzpatrick VI corresponds with the darkest skin.

I: Pale white skin, blue/hazel eyes, blond/red hair, always burns, does not tan
II: Fair skin, blue eyes, burns easily, tans poorly
III: Darker white skin, tans after initial burn
IV: Light brown skin, burns minimally, tans easily
V: Brown skin, rarely burns, tans darkly easily
VI: Dark brown or black skin, never burns, always tans darkly

At PromoCell, we have knowledge of the patients’ skin color (white, brown or black skin), color of eyes and hair, but we don't have any details about the burning/tanning abilities. We therefore classify our tissue donors as follows:

Light (comprising phototypes I and II)
Moderate (comprising phototypes III and IV)
Dark (comprising phototypes V and VI)

Information on the phototype is available for most cell lots isolated from juvenile or adult skin.

Short description: Unpack the box and place the T25 flask(s) in the incubator for 3 hrs (closed cap). Then check confluency under the microscope. When the density is < 70%; aspirate the medium using sterile conditions and add 5-10 ml of the appropriate Growth Medium. The cells should be subcultured according to the subcultivation protocol given in the cells' Instruction Manual once they have reached > 70 % confluency.

1) For fluorescence detection (fluorometer): Black plates with clear bottoms; often clear plates will suffice 2) For luminescence detection (luminometer): White/opaque plates 3) For colorimetric detection (photometer): Clear plates

PromoCell Preadipocytes are isolated from subcutaneous or visceral fat. Subcutaneous fat is found just underneath the skin and it is not related to the obesity-linked diseases. Its accumulation represents the normal physiological buffer for excess energy intake. When the storage capacity of subcutaneous fat is exceeded or the generation of new adipocytes is impaired; visceral fat starts to accumulate. It is located in the abdomen and around internal organs (e.g. kidney; heart; or bladder) and it is linked to hypertension; diabetes; and cardiovascular disease. Adipocytes from subcutaneous and visceral fat differ in several functions; like their response to insulin and other hormones or their lipolytic activity. It highly depends on the scientific problem being addressed; which of the two cell types are best suited for your experiments.

FCS is a natural product consisting of many different components like salts hormones vitamins trace elements proteins and enzymes. There can be large lot-to-lot variations between different serum batches regarding the concentrations of growth-promoting factors. The higher the serum content in a culture medium the higher the impact of the variations on the cell culture system. For this reason PromoCell has developed several serum-reduced and serum-free media where part of the serum has been replaced by more defined factors like cytokines hormones or vitamins.Media with reduced serum concentrations have the benefit that they produce more standardized culture conditions over a long time span.

Please find attached a trouble shooting guide to identify possible reasons for poor detachment during subculture.

Poor attachment after thawing can be a result of inappropriate freezing storing or thawing the cells as well as from inadequate culture conditions (medium incubator). The attached trouble shooting guide should help you to identify the possible reasons.

Yes it is possible to transfect our normal human cells. In general primary and normal cells are much harder to transfect than immortalized cell lines. Apart from the cell type successful transfection also depends on the culture's age and density at transfection the vector used the purity of the nucleic acids the composition of the transfection medium and the experimental conditions.

Both trypsin and accutase represent mixtures of different proteolytic enzymes. Trypsin is prepared from porcine pancreas accutase from invertebrates. Accutase can replace trypsin for the detachment and dissociation of anchorage-dependent cells from surfaces and can also be used on suspension cells to reduce clumping in preparation for counting. The advantages of accutase over the traditional trypsin treatment are that it is more gentle and less damaging to cells (leading to increased viability) and does not contain any mammalian or bacterially derived proteins. Accutase is more thermolabile than trypsin and usually doesn't require an inactivation step.

For the expansion of hematopoietic progenitors (CD133+ cells; CD34+ cells); PromoCell has developed Hematopoietic Progenitor Cell Expansion Medium XF (C-28021); a serum-free and xeno-free formulation. The medium must be supplemented with either Cytokine Mix E (C-39890) or with the user's own mixture of cytokines. Cytokine Mix E is a ready-to-use mix containing recombinant human TPO; SCF; flt3-ligand; and IL-3. The strong expansion of the progenitor cells in Expansion Medium XF + Cytokine Mix E persists for at least two weeks; resulting in a 200-300x increase of the cell number with only 20-30% differentiated cells. Note: If starting with CD133+ cells; the CD133 marker is getting lost during this expansion step. The resulting cells are D34⁺/CD38^-/CD133^-.

Our HAoAF (C-12380) are isolated from the Adventitia the outer layer of the aorta. The cells have been characterized as fibroblasts by the expression of fibroblast-specific CD90.

The PromoCell quality control procedure includes the cultivation of fibroblasts for 15 population doublings. This is to ensure that the cells can be grown for a minimum of 15 PDs (6-8 passages depending on the split ratio used) but it does not mean that the cells immediately senesce after that point. We don't determine the maximum number of doublings or passages but most NHDF lots will certainly achieve > 20 passages.

HFDPC are isolated from the hair papilla of normal human scalp hair follicles. Hair papilla in the adult hair follicle play a crucial role in the dermal-epidermal interactions that control hair production and in hair growth cycle events. The follicle dermal cells are cryopreserved at second passage and can be cultured for at least 10 population doublings when using PromoCell Follicle Dermal Papilla Cell Growth Medium (Cat. C-26501). Typical population doubling times are between 20-36 hrs. The recommended seeding density after thawing/trypsinization is 5;000-10;000 cells/cm². Using 1:4 splits; you can perform 4-5 passages with the cells.

PromoCell's Osteoblast Basal Medium is an optimized media formulation developed for human osteoblast culture. The exact composition is proprietary.

Our subcutaneous HWP are isolated from subcutaneous fat of different localizations; e.g. abdomen; breast; or upper arm. The visceral preadipocytes are isolated from fat surrounding e.g. the pericardium; or from the omentum or mediastinum. The exact localization is specified in the Certificate of Analysis. If you need HWP from a particular localization; please contact our Technical Customer Support prior to placing your order.

PromoCell's Endothelial Cell Growth Medium (C-22010) is a complete medium that can be used for the culture of HUVEC after addition of the SupplementMix. Addition of extra FCS is not necessary. The SupplementMix contains FCS (2% v/v final concentration); recombinant growth factors; hormones; and a bovine brain extract that together have mitogenic effects on endothelial cells.

A subconfluent T25-flask typically contains between 0.9 and 1.2 million cells corresponding to 36;000-48;000 cells per cm². It is recommended to count the existing cell number after trypsinization and to calculate the needed number of new flasks. Recommended seeding density for HUVEC is 5;000-10;000 cells/cm². This usually corresponds to a split ratio of 1:4-1:6. 1:6 means that you can increase the culture surface by factor 6 (e.g. from 1x T25 to 6x T25 or 2x T75).

It actually depends on the culture conditions whether the cells remain in suspension or attach to the surface. When grown in our serum-free; xeno-free HPC Expansion Medium XF (C-28021); the cells remain in suspension.

You can use our standard DetachKit (C-41200) to subculture the human nasal epithelial cells. Some customers still prefer to use our DetachKit-2 (C-41202) as it has a lower trypsin/EDTA concentration but tests in our cell culture lab haven't revealed any adverse effects when using C-41200.

PromoCell's HDLEC are isolated from skin (dermis). When isolated from juvenile donors (C-12216); the exact localization is foreskin. When derived from adult donors (C-12217); the localization depends on the type of surgery; e.g. breast or temple. You can find the information on the exact localization in the Certificate of Analysis. Our HDLEC are tested to be positive for CD31; podoplanin; and Prox-1 and are delivered in P2. The recommended culture medium is Endothelial Cell Growth Medium MV2 (C-22022).

No we don't determine the ratio of HDLEC and HDBEC in our HDMEC lots. From our experience the percentage of HDLEC is highly lot-dependent and can vary between 5 and 60%.

Epidermal Growth Factor (EGF) and hydrocortisone have been reported to have a synergistic effect on the growth of microvascular endothelial cells. Accordingly if you remove the hydrocortisone the proliferation of HDMEC is clearly affected.

Generally you can split differentiated adipocytes. But these cells lose their ability to proliferate after differentiation and you can't expand the culture any more. Therefore it is recommended to plate the cells into the needed vessels (e.g. multiwell plates) prior to induction of differentiation so that trypsinization isn't necessary.

The medium supplemented with Cytokine Mix E is stable for 2 weeks if stored protected from light at 2-8°C.

Soluble receptors are post-translationally modified and generally contain disulfide bonds and glycosylation sites. If produced in E. coli the proteins don't show any biological activity.

Our Human Mesenchymal Stem Cells and Human Pericytes are cryopreserved at the end of secondary culture (P1). After thawing they are in P2.

Serum turbidity is usually caused by cryoprecipitation of lipid components during freezing and thawing. The more times the serum is subjected to freeze/thaw cycles the more turbidity is noticed. It can be minimized by freezing the serum in aliquots at -20°C and thawing these aliquots individually at the time of use.

In principle all microvascular endothelial cells should be able to migrate proliferate and form tubes or sprouts in an appropriate assay after angiogenic stimulation. As this is not part of our routine quality control procedure we cannot tell for sure whether all cell lots will respond to angiogenic stimuli. However PromoCell also supplies HDMEC pre-screened (C-12215) that are especially tested for a positive VEGF response.

These cells are frozen at the end of 2^nd culture. Thawing and seeding results in passage 2 (3^rd culture). We recommend that they be used for differentiation experiments not later than passage 5. The differentiation potential of hMSC in vitro is reduced with ongoing population doublings; meaning the earlier differentiation is induced; the higher the differentiation rates.

PromoCell provides two types of Renal Epithelial Cells: Human Renal Epithelial Cells (HREpC) and Human Renal Cortical Epithelial Cells (HRCEpC). HREpC are isolated from the adult kidney and stain positive for cytokeratin. They comprise a heterogeneous population of renal epithelial cells. HRCEpC are isolated from the cortex of the kidney and comprise cells from proximal and distal tubuli. They also stain positive for cytokeratin.

Short protocol:

Trypsinize the cells as usual
Centrifuge and resuspend in suitable cold freezing medium at a density of 1-4 x 10⁶ cells/ml
Cool down the cells slowly to -80°C (approx. -1°C per min). We recommend to use "Mr. Frosty" from Nalge or "CoolCell" from Biocision; which both provide gradual and controlled cooling rates when placed in a -80°C freezer overnight.
Transfer the vials into liquid nitrogen for long-term storage

For efficient differentiation of our SkMC into myotubes we recommend to use cells that have undergone a maximum of 4-5 population doublings i.e. not more than 1 additional subculturing step after thawing the original vial. For more details about differentiation please see the instruction manual of our Skeletal Muscle Cell Media.

Yes; the Skeletal Muscle Cell Growth Medium can also be used for rat; mouse and rabbit SkMC. We recommend to use the medium right after isolation. Cells that were isolated and cultured in a different medium beforehand may have adapted to the other medium. An abrupt medium change causes stress to the cells resulting in reduced growth rates and lower differentiation capacities.

Our skeletal muscle cells are mainly isolated from M. pectoralis; sometimes also from M. gastrocnemius; M. intercostales or M. gluteus maximus. The exact localization is specified in the Certificate of Analysis.

Short protocol:

Wash the cells with sterile PBS or HepesBSS
Add undiluted accutase to the culture vessel (2 ml per 25 cm²)
Incubate at room temperature for 5-15 min or at 37°C for faster detachment
When the majority of the cells has detached; centrifuge the suspension and resuspend the pellet in fresh medium. In most cases no additional washes or neutralization steps are required.

A concentration > 10% FBS is needed to completely inactivate the trypsin. As most of PromoCell's growth media are serum-reduced or serum-free; the use of a trypsin inhibitor like TNS is highly recommended.

We recommend to use the trypsin as well as the other detach solutions at room temperature to avoid overtrypsinization and irreversible cellular damage.

Our TNS solution contains 0.05% (w/v) trypsin inhibitor from soy bean in HepesBSS/0.1% BSA.

The practice of heat inactivation was originally developed when only serum from adult animals was available. Adult serum contains high serum complement which may destroy cells under certain conditions. Heating serum (30 min 56°C) is intended to inactivate the complement. Today serum is often heat-inactivated without any evidence of beneficial effect. When using FCS (fetal calf serum) heat inactivation is not necessary for most cell lines or cell types. PromoCell does not use heat-inactivated FCS for the preparation of their growth media.

The DMSO concentration in our Cryo-SFM is 10%.

Note: The sale of Cryo-SFM was discontinued at the end of 2024. Cryo-SFM was replaced by Cryo-SFM Plus.

If antibiotics are deemed necessary the recommended final concentrations are: 100 U/ml penicillin + 100 µg/ml streptomycin or 50 µg/ml gentamicin + 50 ng/ml amphotericin B Please note: Addition of antibiotics can reduce the growth rate of the cells.

After addition of the SupplementMix or SupplementPack to our basal medium do I have to add FCS you obtain the complete growth medium. No further supplementation with serum or growth factors is required. Please note: Our media do not contain antibiotics. If you wish to use antibiotics you can add penicillin/streptomycin or gentamicin/amphotericin B at standard concentrations. Addition of antibiotics can however reduce the doubling time of the cells up to 30-40%.

Yes you can aliquot the SupplementMix upon delivery and freeze down 2 or 4 individual aliquots at -20°C. This way you can prepare smaller volumes (2 x 250 ml or 4 x 125 ml) of complete culture medium and thus extend the time you can use the medium.

Upon arrival the basal media should be stored between 4°C and 8°C the supplements at -20°C.

Please do not freeze our cell culture media. Freezing can lead to irreversible precipitation of media components and the quality can no longer be assured.

The lead time is usually 4-8 weeks.

At manufacture ECGS is adjusted to a protein content of 3 mg/ml. For Human Endothelial Cells and Microvascular Endothelial Cells the optimal concentration of ECGS is 2 ml/500 ml medium corresponding to 6 mg extracted protein/500 ml medium. ECGS/H is additionally supplemented with 22.5 mg/ml heparin corresponding to a final concentration of 45 mg heparin/500 ml medium.

The qualitative and quantitative composition of the supplements can be found on our website and in the data sheets of the specialized media. When there is no such information specified; the composition of the supplements is confidential.

The formulation of our basal media is proprietary information. If you need to know the concentration of a particular component for your experiments please contact the PromoCell Technical Customer Service.

It has been shown that phenol red has estrogenic properties. Phenol red-free media are therefore generally used in studies evaluating steroid hormone action in cultured estrogen-responsive cells (Berthois et al. 1986). Furthermore phenol red can also interfere with some analytical methods like photometric analyses.

The Growth Medium Kit allows customization of the end concentrations of growth supplements. It is therefore more flexible than the Medium "ready-to-use" and can eg. be used to prepare a starvation medium. Please note: Modification of supplement concentrations may have an impact on cell growth. You should test in advance whether and for how long the cells can survive the altered culture conditions.

Our DetachKit (C-41200) is optimized for primary cells and can be used to trypsinize all Normal Human Cells supplied by PromoCell. Detach Kit-2 was developed for very gentle detachment and is still used by some investigators to subculture Human Nasal Epithelial Cells (HNEpC).

Both Kits contain HepesBSS trypsin/EDTA and Trypsin Neutralizing Solution (TNS). In DetachKit (C-41200) the concentration of trypsin/EDTA is 0.04% / 0.03%. In DetachKit-2 (C-41202) it is reduced to 0.025% trypsin / 0.01% EDTA. HepesBSS and TNS concentrations are identical in both Kits.

The three components of DetachKit and DetachKit-2 (HepesBSS Trypsin/EDTA TNS) are stable for 1 year from the date of manufacture when stored at -20°C. Once thawed for usage they should be stored at 4-8°C and can be used for 6 weeks. Please avoid repeated freeze/thaw cycles.

Our Normal Human Cells have been cultured and tested in our growth media and have adapted to these conditions. Using other media may yield unsatisfactory results due to suboptimal supplies of nutrients and growth factors. PromoCell can only guarantee good cell growth (as stated in the Certificate of Analysis) when the cells are grown in the recommended media.

The human MSC derived from bone marrow adipose tissue and umbilical cord matrix are from different origins but with comparable biological properties and function. Depending on the tissue of origin they may have a higher preference for differentiation into one particular cell type and a lower preference for another one but they all still retain the differentiation potential for the mesenchymal lineage.

For optimal cell growth the human CD34⁺ Progenitors (C-12921) are cultured in PromoCell Hematopoietic Progenitor Cell Expansion Medium XF (C-28021) supplemented with Cytokine Mix E (C-39890) or with any other cytokine cocktail suitable to achieve optimal cell expansion. Cytokine Mix E contains recombinant human TPO SCF flt3-ligand and IL-3. The strong expansion of CD34⁺ cells will persist for approx. 2 weeks. The expansion factor is usually between 75 and 200-fold.

During the isolation of our Human Mononuclear Cells (hMNC) we first of all pay attention to thoroughly discard the platelet-containing fraction before we aspirate the MNC containing-interphase of the Ficoll gradient. The harvested MNC fraction is then subjected to several washing steps to remove potential remaining platelets. Finally the cell preparations are verified by microscopy to be largely free of platelet contamination.

There is a strong variation from donor to donor and sometimes from donation to donation concerning the number of vials that can be produced. This is due to individual variances between the donors as some of them have more MNCs per ml of blood and others have less. Generally; the number of vials (each with 25 x 10⁶ cryopreserved cells) per lot ranges between 10 and 40.

Pericytes have been shown to differentiate e.g. into adipocytes osteoblasts chondrocytes fibroblasts/myofibroblasts vascular smooth muscle cells and phagocytes.

CD146 (MUC18) is a surface marker expressed on pericytes MSCs and endothelial cells from large vessels (but not microvascular endothelial cells). In combination with CD34 (a marker for endothelial and hematopoietic cells) pericytes can be characterized by FACS analysis as CD146⁺/CD34^-. The absence of CD34 expression makes sure that the cells are not of hematopoietic/endothelial origin. Further markers that have been described for pericytes are NG2 CD90 alpha-SMA and PDGFR-beta.

Our kidney cells are characterized by their epithelial cell morphology and by cytokeratin expression using a pan-cytokeratin antibody.

Our human pulmonary microvascular endothelial cells (C-12281) are sourced from the lung parenchyma with all large vessels being removed beforehand. Therefore most of the HPMEC originate from capillaries.

Our HPF (C-12360) are isolated from peripheral lung tissue.

All cytokines in Cytokine Mix E (human TPO SCF flt-3 ligand and IL-3) are produced in E.coli. They are purified by chromatography are free of endotoxins and are tested for their biological activity.

Alcian Blue stains the extracellular matrix of chondrocytes; e.g. cartilage-specific aggrecan and other glycosaminoglycans. To our knowledge; chondrocytes only express aggrecans when grown in 3-D culture and not in 2-D culture. To detect cartilage specific markers in monolayer culture; it is recommended to perform immunofluorescence detection of collagen type II.

Human Mesenchymal Stem Cells have retained the ability to differentiate into a variety of cell types including fat cells; chondrocytes; and osteoblasts. PromoCell supply hMSC from 3 different tissues: bone marrow (hMSC-BM; C-12974); umbilical cord matrix (hMSC-UC; C-12971); and adipose tissue (hMSC-AT; C-12977).

MSC-BM show very good differentiation into bone cells but also into chondrocytes and fat cells when the respective Differentiation Media is used.
MSC-UC have a high potential to differentiate into chondrocytes but only weak potential for fat or bone cell differentiation.
In contrast; MSC-AT differentiate very well into fat and bone cells but only moderately into chondrocytes.

In other words; to obtain a high percentage of bone cells; MSC-BM or MSC-AT are the cells of choice. There are of course lot-to-lot variations and the differentiation will decrease from passage to passage. If you need MSCs with a particularly high osteoblast differentiation capacity; you can call our Technical Customer Service before placing your order so that we can select an appropriate cell lot for you.

Adipogenic differentiation can be identified morphologically and without any staining by the formation of intracellular lipid vesicles.
In contrast; when MSC differentiate into bone cells; there is no significant change in morphology. It is recommended to perform Alkaline Phosphatase staining to detect osteoblastic differentiation or Alizarin Red S staining to show osteoblast mineralization.
Chondrogenic differentiation is generally performed as spheroids in 3-D cell culture and not in 2-D monolayer culture. Staining with Alcian Blue to visualize the differentiation process is indispensable.
Neurogenic differentiation can be detected using neuron specific markers (e.g. beta-3 tubulin; NeuN; MAP2) and by their typical neuronal morphology.

For differentiation protocols; please see attached Application Notes.

Our hMSC-AT are characterized by their differentiation potential into chondrocytes; fat cells; and bone cells. In addition; we determine the presence of CD73; CD90 and CD105 expression as well as the absence of CD14; CD19; CD34; CD45 & HLA-DR expression by flow cytometry as proposed by the International Society for Cellular Therapy.

Mononuclear cells are mostly used in immunology; infection biology; hematology and cancer research to study subpopulations of blood cells. Our Mononuclear Cell Medium (C-28030) is intended for short-term maintenance (up to 48 hrs) of the thawed hMNC before you proceed with your experiments. The number of PDs will depend on the subsequent cell culture conditions and is not determined by PromoCell. Please note: Depending on the conditions; the ratio of the subpopulations will gradually change; as the different blood cell types behave in different manners. Researchers normally start soon after thawing to either select the cell type of their interest (e.g. hematopoietic cells; endothelial progenitor cells) or perform experiments with all populations of hMNCs (e.g. to study effects on toxicity; viability or metabolism).

We have tested the differentiation capacity of our hMSC into adipocytes chondrocytes and osteoblasts over time and still see good differentiation rates after 10 population doublings i.e. at passage 5. However the differentiation potential declines with ongoing population doublings. To obtain optimal differentiation rates experiments should be performed as early in culture as possible.

For chrondrogenic differentiation it is important that the cells do not adhere to the wells. During differentiation the cells form spheroids which float in the medium. Therefore there are no special requirements for the wells as long as they are U bottom shaped and suitable for suspension culture.

The following criteria are applied for blood donations: a) Donors with light infection like a cold or cough are excluded for one week b) Donors with infections with a temperature above 37.9°C and/ or antibiotics therapy are excluded for 4 weeks c) Blood pressure: exclusion only if systolic blood pressure is < 100 mm Hg or > 180 mm Hg or if diastolic blood pressure is > 100 mm Hg. No exclusion if blood pressure is drug treated and in acceptable range. d) High cholesterol: no exclusion even though medication is used e) Diabetes: exclusion if diabetes type I or use of insulin f) Steroid use: exclusion for 4 weeks after application g) Cancer: exclusion h) Other chronic diseases: exclusion depends on the type of disease (for example chronic heart disease; autoimmune disease)

We have many customers who perform starvation with our HUVECs. Most of them use Endothelial Cell Basal Medium supplemented with FCS (0.5-1% for shorter periods; 5-20% for 24-48 hrs). The cells have to be in a good condition and the experiment should be terminated soon after starvation. Prolonged periods will induce apoptosis.

Yes; it is possible. Short protocol: Plate human osteoblasts in Osteoblast Mineralization Medium on collagen I coated TC vessels. Incubate the cells for 17-21 days and change the medium every third day. Be careful not to disturb the cell monolayer. Fix the cells. The calcium deposition can be visualized by von Kossa or Alizarin Red staining. More detailed information on osteoblast mineralization and Alizarin Red S staining can be found in the attached Application Note.

PromoCell guarantee > 15 PD for their HUVEC. The number of passages that can be performed; depends on the dilution factor used during subculture. If you split the cells 1:4; they can perform about 2 population doublings per passage which means that they can be cultured for at least 6-8 passages.

All HDMEC pre-screened lots that we currently have in stock are isolated from foreskin. If you need pre-screened HDMEC from adult donors; please contact the PromoCell Technical Customer Service.

The standard medium for isolation and propagation of our HUVEC HUAEC HPAEC and HSaVEC is Endothelial Cell Growth Medium (C-22010). It contains ECGS an extract from bovine hypothalamus which has mitogenic effects on endothelial cell proliferation. Scientists who prefer a more defined Growth Medium can use Endothelial Cell Growth Medium 2 (C-22011). In this medium ECGS is replaced by VEGF IGF and additional bFGF and EGF to stimulate endothelial cell growth.

Our HCMEC (Human Cardiac Microvascular Endothelial Cells) are not endocardial cells. They are isolated from the capillaries in the heart muscle. Therefore they are in fact microvascular.

Our juvenile HDMEC (C-12210) are isolated from foreskin of young male donors (1-10 years). In contrast; adult HDMEC (C-12212) are derived from different skin localisations like the cheek; temple; or breast. The donors are > 20 years old and are mostly female. Adult HDMEC are the cells of choice when you need cells from a particular part of the body (other than foreskin); or if it is important for your study to use cells from female and/or adult donors.

Our chondrocytes are derived from patients (~55-80 years) who underwent surgery for total endoprothesis of the hip or knee joint. In most cases this is necessary due to arthrosis. If the tissue shows macroscopic lesions it is not used for cell isolation.

PromoCell generally culture their HOB on uncoated tissue culture dishes. It is possible however to grow them on collagen type I- or fibronectin-coated dishes as well. Please note: The type of extracellular matrix used may influence the expression of certain genes (e.g. integrins) and thereby affect cellular metabolism. Therefore we recommend to always use the same type of coating matrix for a whole set of experiments.

PromoCell Growth Media have special formulations and are much more complex than DMEM or RPMI + FBS. In comparison to immortalized cell lines primary cells have much higher nutrient and growth factor requirements. Classical media do not usually achieve good performances with our cells.

All Adult Human Blood and Stem cells are analysed by flow cytometry to express defined markers. MSCs are additionally tested for their capacity to differentiate into the osteogenic; chondrogenic and adipogenic lineages. For more details; please see Certificate of Analysis of the respective cell type ("Phenotypic characterization").

It is not necessary to use coated flasks for (most of) our Normal Human Cells but it can be done. As coating with extracellular matrix proteins can affect cellular metabolism; it is recommended to use the same coating material for a complete set of experiments. Cells that need to be grown on coated dishes:

Mesenchymal Stem Cells (C-12974/C-12971/C-12977) need Fibronectin-coating when grown in PromoCell MSC Growth Medium XF (C-28019) and when differentiated in MSC Neurogenic (C-28015); Adipogenic (C-28016); or Osteogenic (C-28013) Differentiation Media.
Human monocyte-derived macrophages (C-12914/C-12916/C-12915/C-12917) must be seeded into Fibronectin-coated culture vessels in combination with PromoCell's M1- and M2-Generation Media XF (C-28055; C-28056).
For efficient induction of osteoblast mineralization with PromoCell's Osteoblast Mineralization Medium (C-27020); the TC plates should be pre-coated with collagen type I.

The population doubling time or generation time (t_g) is usually calculated during the logarithmic phase of growth. It specifies the time (t) in hours needed by the culture to double its cell number. t_g = t / n n: number of population doublings

PromoCell cell pellets are made from > 1 million cells and are dissolved in 200 µl RNAlater.

The majority of PromoCell cell pellets (C-14**) are prepared 1 passage after thawing the cryopreserved cells. Examples: HUVEC and HUAEC correspond to P1 after thawing; therefore the pellets are frozen in P2. SMC or keratinocytes or epithelial cells correspond to P2 after thawing; therefore the pellets are frozen in P3. In contrast; our blood cells are cryopreserved directly after cell isolation. These pellets are prepared after thawing the cryovials with no further cultivation step.

The recommended media for a particular cell type are specified on the respective product page ("Recommended Products") and can be found in the Manual belonging to the cells.

The recommended Growth Media are specified in the Manual belonging to the cells and can be also found on the product page of the cells ("Recommender products").

Yes you can use accutase to detach Normal Human Cells. Accutase acts very gently on the cells. Cell membranes and surface epitopes will not be harmed. It is therefore mostly used for applications that require unchanged surface markers e.g. for flow cytometry or for detachment of very sensitive cells.

PromoCell recommend to trypsinize all Normal Human Cells at room temperature and to monitor the detachment under the microscope. Prolonged trypsinization at 37°C can lead to irreversible damage of the cells.

Yes but the experimental starvation conditions have to be determined individually for each cell type. Usually the cells are maintained in basal medium with reduced growth factor concentrations or lower FBS content. The cells need to be in a good condition and the starvation should be kept as short as possible as prolonged serum and/or growth factor deprivation induces apoptosis.

Most commercially available trypsin solutions have trypsin concentrations of 0.05% or higher which can harm the primary cells. PromoCell recommend using a 0.04% trypsin / 0.03 % EDTA solution for the subculture of Normal Human Cells.

The PromoCell trypsin 0.04% / EDTA 0.03% solution and the PromoCell TNS solution are both based on HepesBSS. Therefore it is best to also use HepesBSS to wash the cells prior to trypsinization.

We recommend to use the DetachKit when subculturing our Normal Human Cells. It contains a HepesBSS washing buffer; trypsin 0.04% / EDTA 0.03% solution; and TNS; a trypsin inhibitor from soybean. Please note: Many of our culture media have low serum content or no serum at all. These media are not suitable to inactivate trypsin during subculture.

Yes RNAlater Solution will denature proteins. Therefore protein obtained from PromoCell cell pellets will be suitable for applications such as Western blotting or 2D gel electrophoresis but not for applications that require native protein.

There are two options for isolating RNA from cells stored in RNAlater Solution: 1) The solution is removed from the cells prior to extraction by centrifugation at 5;000 x g for 10 minutes at 4°C. Note: Because of the density of RNAlater® solution; greater centrifugal forces are required to spin down the cells. 2) If no pellet is visible after centrifugation; RNA can also be purified directly from the RNAlater® solution. This can be done by adding 2 ml of 10x lysis buffer; and proceeding normally.

PromoCell cell pellets (C-14**) are an easily accessible source of DNA RNA and proteins.

No the PromoCell cell pellets (C-14**) are frozen at -20°C and cannot be revived. Their main application is to analyze RNA or protein. Cryopreserved cells that can be revived are available from the same donors. Please contact our Technical Customer Support if you need matched viable cells.

Cell type-specific markers are usually determined one passage after thawing. Example: After thawing; HUVEC are in P1. Markers (CD31 expression; Dil-Ac-LDL uptake) are tested in P2. Please note: Cells that are frozen directly after isolation (blood cells) are tested directly after thawing with no further culturing step.

a) HNEpC (Human Nasal Epithelial Cells) are isolated from nasal mucosa b) HTEpC (Human Tracheal Epithelial Cells) from the surface epithelium of trachea c) HBEpC from the surface epithelium of bronchie; and d) HSAEpC (Human Small Airway Epithelial Cells) from the distal portion of the respiratory tract in the 1 mm bronchiole area (comprising the cells from bronchioli and alveoli).

HDLEC and HDBEC both express the typical endothelial cell marker CD31 (= PECAM-1).
HDLEC cultures are additionally tested positive for podoplanin; a transmembrane glycoprotein involved in lymphatic vessel formation; whereas HDBECs are podoplanin-negative.

Our HUtMEC are isolated from the middle layer of the uterine wall (myometrium). The tissue donors were not pre-treated with hormones.

Our HPMEC are isolated from peripheral lung tissue of adult donors.

Juvenile HDMEC (C-12210) are isolated from the dermis of foreskin. Adult HDMEC (C-12212) are isolated from different regions. The localizations include cheek temple breast upper arm and labia. Please contact our Technical Customer Support if you need HDMEC from a particular part of the body.

Our Chondrocyte Growth Medium consists of an optimized basal media formulation and is supplemented with 10 % (v/v) fetal calf serum that has undergone stringent biological controls.

PromoCell cell pellets (C-14**) can be stored indefinitely at -20°C and are stable for up to 1 month at 4°C and up to one week at room temperature. Please note: The samples do not freeze at -20°C.

The passage number varies depending on the cell type. Please refer to the Manual for your cells under "Specifications". You will also find the information in the Certificate of Analysis (CoA).

The recommended plating density after thawing/subculture may vary depending on the cell type. It is specified on the Website (tab: additional Information) and in the Product Manual.

The recommended plating density after thawing/subculture may vary depending on the cell type. Please refer to the data sheet for your cells under "Specifications".

The Certificates of Analysis can easily be downloaded from our PromoCell website: https://www.promocell.com/certificate-of-analysis/ Simply type in the lot number indicated on the cryovial/TC-flask and click the SEARCH button.

In principle; both types of liquid nitrogen storage are acceptable; each having its advantages and disadvantages.

Liquid phase storage provides a consistent temperature of -196°C; a longer holding time and a greater vial capacity but involves the risk of contamination issues.
Storage in the gas phase is very safe with respect to contaminations but the holding time of the cells is shorter and the vial capacity is reduced.

PromoCell guarantee 15 population doublings (PD) for most Normal Human Cells (unless otherwise indicated on the Certificate of Analysis) when the recommended PromoCell media and the PromoCell DetachKit are used. For more information; please check the respective Manual; section "Specifications".

PromoCell does not determine the number of passages but instead we calculate the population doublings (PD) that can be performed with the cells. The term passage only describes the process of detachment and replating and does not take into account different split ratios. The optimal split ratio is calculated from the actual cell yield after trypsinisation and the recommended plating density. In most of our cell types; the split ratio is usually between 1:3 and 1:6. Using 1:4 splits (i.e. increasing the growth surface by factor 4 each time); 15 doublings are achieved after 6-8 passages. For recommended plating densities; please view the respective Manual; section "Specifications".

"Adult stem cells" are stem cells isolated from postnatal tissues that have retained the capacity for cell renewal as well as for differentiation into multiple lineages (multipotency).

Freshly isolated cells that are plated in a tissue culture vessel for the first time are named primary cells or primary culture (corresponding to P0). As soon as they have been subcultured; they should correctly be termed normal cells (> P1).

Generally; the medium should be changed every 2-3 days. Please note: Following thawing; the first medium change should be performed after 16-24 hours to prevent cell damage due to residual freezing medium.

Our Osteoblast Supplement consists of FCS (10 % [v/v] final concentration) which is specifically tested to support optimal growth of Normal Human Osteoblasts.

General protocol for recovery of anchorage-dependent primary cells: Remove vial from liquid nitrogen; transport it on dry ice to the cell culture lab. Thaw in a 37°C waterbath for approx. 2 min; until it is just defrosted. Keep the vial immersed in water until just below the screw cap during thawing and only remove it shortly after approx. 90 sec to check the progress. Do not repeatedly insert and remove the vial while thawing in water. Carefully disinfect the vial with plenty of 70% EtOH under the laminar flow hood and aseptically transfer the thawed suspension into an appropriate TC dish with growth medium (pre-warmed in the incubator for > 30 min). The cells usually attach within a few hours. Perform media change after 24 hrs at the latest to remove residual DMSO from the freezing media. Additional information can be found in the instruction manuals of our cells.

PromoCell Cell Culture Media "ready-to-use" consist of basal medium and SupplementMix. PromoCell Culture Media Kits consist of basal medium and SupplementPack. Addition of the supplements (SupplementMix or SupplementPack; respectively) to the appropriate basal medium will result in identical growth media.

Your observation can be either ascribed to a change of the culture medium to a cross-contamination with another cell type or to differentiation or senescence. The attached trouble shooting guide should help you to identify the reason for your observation.

Microbiological contaminations can be introduced into cell cultures by unsterile working techniques contaminated water baths media plasticware etc. Please check the attached trouble shooting guide for more detailed information.

Normal cells have a finite life span and therefore eventually stop growing and become senescent. In addition a cease in proliferation or cell death can be induced by other factors. For more details check the trouble shooting guide below.

Slow growth after subculture can be caused by over-trypsinization or other suboptimal culture conditions. Please see attached trouble shooting guide for possible reasons.

Several different methods for the detection of mycoplasmas have been described like e.g. cultures on agar in liquid or semi-solid media staining with DAPI mycoplasma-specific antibodies biochemical methods and PCR-based assays. PCR-based detection is very sensitive detects all mycoplasma species that occur in cell cultures and is completed within 3-5 hours.

At PromoCell; we get the umbilical cords from our tissue suppliers with no addition of buffers or media. This method prevents the microorganisms from being washed into the blood vessels. Before we start the cell preparation; the umbilical cord is also cut on both ends with a sterile scalpel to provide sterile intersections in addition to the sterile lumen. This method allows us to isolate sterile endothelial cells from umbilical vein and to plate them in antibiotics-free culture media.

Typical cell densities are between 32.000 - 40.000 cells/cm² (approx. 800.000 - 1 million cells per T25-flask).

For mineralization assays; HOB are cultured in Osteoblast Mineralization Medium (C-27020). Mineralization can be detected after approximately 3 weeks by incorporation of Ca-45; or it can be visualized by von Kossa or Alizarin Red staining for calcium.

If you purchase a proliferating culture of human osteoblasts (C-12760); there will be > 500;000 cells in the TC-flask. Once the culture is subconfluent; you will count between 750;000 - 1.1 million cells/T25 (depending on the cell lot).

Normal osteoblasts similar to other non-transformed cell types can be expanded in vitro to a certain extent before they are used for experiments. Nonetheless HOB are generally used at low passages (up to P4) in most labs.

Our Osteoblast Growth Medium (C-27001) consists of the Basal Medium supplemented with 10% (v/v) FCS but with no recombinant growth factors. The medium primarily supports the proliferative capacity of normal human osteoblasts. It does not contain osteogenic factors (like dexamethasone and beta-glycerophosphate) that promote differentiation as many users test their own chemical compounds (growth factors hormones) or examine the effects of physical strain or sheer stress on the differentiated functions. The Osteoblast Growth Medium is well-suited as the basis for these applications and can be supplemented with further growth factors if necessary. To specifically induce mineralization PromoCell supply Osteoblast Mineralization Medium (C-27020).

Induction of differentiation: - Grow the cells in Preadipocyte Growth Medium (C-27410) until they reach complete confluence; this roughly takes 1 week. Change medium 2-3x per week. - Aspirate the Growth Medium; add Preadipocyte Differentiation Medium (C-27436) for 72 h - Aspirate the Differentiation Medium; add Adipocyte Nutrition Medium (C-27438). The cells will now start to accumulate droplets of lipids which can be visualized under the microscope. The process usually takes 1-2 weeks. Change medium 2-3x per week. It is possible to maintain the differentiated adipocytes in Nutrition Medium for up to 4 weeks. However; the cells tend to lyse and detach after about 3 weeks. Please note: We recommend to perform differentiation experiments at population doubling numbers lower than 4-5 (a maximum of 1 passage after thawing) in order to obtain a high differentiation level of the culture.

Yes our Skeletal Muscle Cell Differentiation Medium is completely defined. The SupplementMix (C-39366) consists of recombinant human insulin.

PromoCell Fibroblast Growth Medium is serum-free and therefore not subjected to the lot-to-lot variations observed with DMEM/10% FCS. Our Fibroblast Growth Medium therefore allows for much more standardized cell culture conditions.

After adding the Skeletal Muscle Cell Differentiation Medium; myoblasts will start to differentiate into myotubes and stop growing. The cells should not be split anymore. It is recommended rather to plate the SkMC into the needed vessels; (e.g. multiwell plates); prior to induction of differentiation and to perform studies directly on differentiated cells. If it is necessary for your tests to detach the myotubes and they are difficult to trypsinze; you can use a "rubber policeman".

Our SkMC are proliferating myoblasts that have retained the capacity to differentiate. Upon withdrawal of serum and growth factors differentiation is induced and the cells form multinucleated syncytia.

The PromoCell Basal Media must be stored between 4-8°C and should not be frozen; as this can lead to precipitations. The same is true after addition of the supplements: the complete medium has to be kept at 4-8°C. If you prefer to make up smaller volumes of complete medium; you can aliquot the Supplement Mix and refreeze those aliquots at -20°C until use. This way you can extend the period in which you can use the supplemented media.

We have obtained best results when the cells have reached 60-80 % confluency. At this point the Growth Medium is aspirated and replaced by Skeletal Muscle Differentiation Medium. After 2 - 8 days extensive formation of multinucleated syncytia can be observed. For a stable differentiation of SkMC switch back to Skeletal Muscle Cell Growth Medium after 5 days incubation in Skeletal Muscle Differentiation Medium.

To differentiate the SkMC into myotubes; we recommend to use cells that have undergone a maximum of 4-5 population doublings. For this; the cells (ideally in P2 or P3) are cultured to 60-80% confluency in Skeletal Muscle Cell Growth Medium (C-23060). Then; a change to (serum-free) Differentiation Medium (C-23061) is performed to induce the differentiation process (formation of multinucleated syncytia). After 5 days; switch back to the Skeletal Muscle Cell Growth Medium for another 8 days to complete the differentiation. This protocol leads to stable myotubes and some of the myotubes show spontaneous contractions.

Recommended procedure: - Thaw the vial according to our protocol - Seed one part of the cells (5;000 cells/cm²) directly into multiwell plates for subsequent differentiation induction; grow in Preadipocyte Growth Medium until they reach 100% confluency; then induce differentiation according to the recommended protocol - Seed the other part (5;000 cells/cm²) into a TC dish and expand them in Preadipocyte Growth Medium; trypsinize at subconfluence; if necessary divide the cell suspension again into two parts and use one part for differentiation tests (see above); the other part to continue the culture of undifferentiated HWP. - Differentiation capacity may decline after 1-2 passages in vitro; therefore best perform your differentiation tests at early passages. HWP at higher passages can still be used for studies that require undifferentiated HWP (e.g. proliferation assays).

The basic information we receive from the surgeons about the tissue donors usually includes age; gender; and ethnicity.

For many of our donors of subcutaneous preadipocytes; we also have information on BMI; hair color; skin pigmentation; and; in some cases; smoking habits or known diseases (e.g. Diabetes). Most of our subcutaneous HWP donors are between ∼25-65 years old.
The visceral HWP donors are mostly between ∼20-75 years old. For many cell lots we know the BMI; in some cases also the hair color; skin pigmentation; smoking habits; and/or known diseases (e.g. Diabetes or COPD).

If you are looking for particular specifications; please contact our Technical Customer Support; so that we can offer you appropriate HWP lots.

We recommend a seeding density for chondrocytes between 10.000 and 20.000 cells/cm². This means that a subconfluent T25-flask with approx. 900.000 cells/T25 flask (36.000 cells/cm² ) may be either split into 3 new T25 or seeded in one T75 flask or in one 100 mm petri dish. We do not recommend a specific type or brand for the culture of HCH.

Our HPAEC (C-12241) are harvested directly from the pulmonary artery. For their isolation; the vessel is explanted right after the position where the artery leaves the heart; including the bifurcation. HPAEC represent the innermost cell layer (i.e. the endothelial cells) of the pulmonary artery. We also supply pulmonary microvascular endothelial cells (HPMEC; C-12281) isolated from the capillaries of peripheral lung tissue.

The optimal calcium concentration for both proliferation and differentiation of keratinocytes depends on the species and also on the media formulation. To keep primary human keratinocytes in the proliferative status concentrations between 0.03 and 0.15 mM (PromoCell Keratinocyte Growth Medium 2: 0.06 mM) are generally used. Increasing the calcium above 1 mM will induce terminal differentiation and lead to the loss of proliferative activity.

It usually takes 5 to 8 days to grow our Normal Human Chondrocytes (C-12710) to subconfluency. The number of doublings (PDs) they undergo can be calculated from the number of seeded cells and the cell yield at subconfluency. Generally; when HCH are plated with 10;000 cells/cm² they perform between 1.5 and 2 doublings per passage.

The Chondrocyte Growth Medium SupplementMix consists of Fetal Calf Serum (final concentration 10 % v/v) which has specifically been tested for the culture of primary chondrocytes. FCS contains a variety of different growth factors which are however not analyzed in more detail. There are no further growth factors added to the SupplementMix.

Upon arrival; you can either defrost the cells immediately and seed them in a TC vessel or store the vial in liquid nitrogen until needed. It is possible to re-freeze normal human cells but PromoCell does not recommend it; because each freezing cycle leads to a loss in proliferation potential. If you want to freeze them; please use Cryo-SFM (C-29910) which is a serum-free freezing medium that prevents the possibility that any serum is introduced into your serum-free cell culture system. It is also recommended to freeze the cells at an early passage.

We guarantee 15 population doublings for our NHEM. In terms of passages this corresponds to approx. 6-8 subcultivations.

We can however not guarantee that the activity of all melanocyte genes remains unchanged during this time. Primary cells do gradually change their phenotype in vitro. Therefore, it is recommended to use the cells for the important experiments at low passages.

After isolation and purification of our primary human melanocytes, the cells are checked during quality control whether they show a typical morphology and whether they express the marker Mel-5. Mel-5 is a 75 kDa glycoprotein usually expressed by normal melanocytes. Our Melanocyte Growth Medium (C-24010) has been developed to promote melanocyte growth in vitro. It does not however, completely block the growth of other cells (such as: NHEK or NHDF).

Therefore, it is important to have a pure melanocyte culture from the very beginning.

In contrast, the Melanocyte Growth Medium M3 (C-24310) is much more selective and represses the growth of contaminating cells much better.

The majority of our skin tissue donors are caucasians. But occasionally we also get skin biopsies from asian and black donors.

Please contact our Scientific Support if you need cells from a particular phototype or origin. They will check our inventory and send you a list of available cell lots.

PromoCell Chondrocytes can be expanded in normal monolayer culture using our Chondrocyte Growth Medium (C-27101). The Medium consists of an optimized formulation and is supplemented with 10 % FCS. De-differentiation of chondrocytes is a known phenomenon observed during in vitro-culture after a period of approx. 2 weeks; but in vitro-culture is needed to expand the cells. Once a suitable cell number is obtained; the monolayer system can be changed to a more complex 3-D system either by culturing the cells on substrates like alginate beads; gels; or degradable polymer scaffolds or by using 3-D spheroid culture. Using appropriate conditions; re-differentiation is triggered and the cells start producing cartilage-specific ECM again. PromoCell does not supply a special culture system but we use 3-D spheroid culture and Alcian Blue staining to characterize our chondrocytes during quality control. The protocol is very similar to the one we use for chondrogenic differentiation of MSC.

Mature chondrocytes from articular cartilage are postmitotic producers of extracellular matrix components like collagen types II IX and XI and proteoglycans. Upon release from the tissue of origin and seeding in monolayer culture cells re-enter the cell cycle and proliferate. After a period of approx. 1-3 weeks they will gradually start dedifferentiation and will adopt a fibroblastic phenotype. Dedifferentiation can be detected by decreased collagen type II or increased collagen type I expression as well as by the appearence of the fibroblast marker Thy-1/CD90. Re-differentiation can be induced by changing from conventional monolayer culture to a 3-D culture system.

All our chondrocytes (C-12710) are isolated either from knee joint or femoral head. If you need chondrocytes specifically from either localization please contact our Technical Customer Service before placing your order. They will send you the available lot numbers from the localization that you are looking for.

We get 5-8 new lots of adult MNC (C-12907) every month (> 15 vials each). Cord blood MNC (C-12901) are smaller lots (5-10 vials); we get 2-4 new lots every month.

Our MNC lots are checked for the rate of lymphocytes monocytes and granulocytes w/o staining by FSC/SSC using a flow cytometer. A sample plot is attached. For our purified CD14⁺ monocytes we additionally perform a CD14⁺ staining.

The cells will die. Hematopoietic Progenitor Cell Expansion Medium XF always must be supplemented with Cytokine Mix E or an appropriate cocktail of cytokines.

The study of angiogenesis has been significantly advanced by the ability to culture endothelial cells in vitro. Initially large vessel ECs such as those isolated from the human umbilical vein (HUVEC) were used for these studies but increasingly it has been recognized that microvascular endothelial cells are a more appropriate model since angiogenesis involves microvessels rather than large vessel ECs.

PromoCell Normal Human Cells should be cultured in the appropriate medium at 37°C and 5% CO₂ in a humidified atmosphere. Please note: If using cell culture flasks w/o filter cap; unscrew the cap by half a turn to allow sufficient ventilation.

Fibroblast contamination cannot be completely avoided in primary cell cultures. As epithelial cells attach more firmly than fibroblasts it is possible to perform partial trypsinization to remove the fibroblasts. This is done by adding trypsin/EDTA to the TC dish for 2-4 min. When the fibroblasts detach the enzyme is inactivated and the suspension with the fibroblasts aspirated. The remaining epithelial cells are washed twice with buffer and their culture is continued in the respective Growth Medium.

Precoating of culture vessels with ECM proteins does not have adverse effects on the cells but has been reported to influence the cellular expression pattern. It is therefore recommended to use the same culture conditions e.g. fibronectin- collagen- or gelatin-coating for a whole set of experiments to be able to compare the results.

No it is not necessary to use coated flasks therefore we don´t recommend their usage in the Instruction Manual. However for special applications some of our customers use collagen-coated dishes.

We source the bronchial tissue from forensic medicine and from thoracic surgery. For some lots; the smoking habits of the donors are known. Please contact our Technical Customer Service before ordering the cells if you need this information.

Our Nasal Epithelial Cells are isolated from nasal septum or adenoids.

The tissue comes from patients who underwent heart transplantation. We obtain a part of the explanted (not the transplanted) heart to prepare cardiac myocytes.

PromoCell guarantees 15 population doublings (PDs) if the HCM are grown in Myocyte Growth Medium. Depending on the cell lot and the culture conditions; the cells can be maintained in culture for > 6-8 passages corresponding to a period of 1-2 months.

The amount of media needed per vial depends on the growth characteristics of the cells the size of the TC vessels and the split ratios used the frequency of media changes the type of experiments you perform etc. It is therefore difficult to give definite quantities. As a rough guideline 1-2 bottles (500 ml each) are needed for 1 vial of HUVEC.

During our quality control we do not determine the presence or maintenance of respective stem cell markers in the tumorspheres. We use a functional approach instead; by culturing the tumorspheres over serial passages in C-28070. Tumorsphere formation requires the biological features of Anoikis resistance and self-renewal and therefore indicates the presence of cancer stem cells (CSC)/cancer initiating cells (CIC) in the spheres. 3D tumorsphere culture thus allows scientists to study the biology of CSCs without any background knowledge on CSC markers.

Please see our attached alternative product guides for media and cells. Please note: Our media do not contain antibiotics. For optimal cell growth; we recommend to refrain from using antibiotics. However; when a sterile environment cannot be 100% ensured; it may be advisable to add antibiotics to your media so as to protect the cultures from potential microbial infections.

There are several factors that can influence successful transfections; e.g. viability and density of the cells; choice of the transfection reagent; quality and type of the transfected molecules (plasmids; siRNA; oligonucleotides); as well as the culture medium and supplements used. When using PromoCell Endothelial Cell Growth Media for cell transfection; please follow the instructions below: Heparin; which is included in our Endothelial Cell Growth Medium (C-22010/C-22110); Medium 2 (C-22011/C-22111) and Medium MV (C-22020/C-22120) may reduce the transfection efficiency. We therefore recommend to use our heparin-free Endothelial Cell Growth Medium MV2 (C-22022/C-22121). Alternatively; you may use Endothelial Cell Growth Medium Kit (C-22110); Medium 2 Kit (C-22111); or Medium MV Kit (C-22120) without adding the ECGS/heparin supplement to the Basal Medium. Please note: Before and after transfection; the cells should be cultured in complete Growth Medium including heparin to ensure optimal growth.

Yes; we have received a customer feedback that our MSC Growth Medium 2 also works for rat MSCs. The rat cells grow nicely in this medium and have a good viability.

The following cell lines have been tested at PromoCell to form tumorspheres in 3D Tumorsphere Medium XF (C-28070):

U-87 MG
MCF-7
MDA-MB-231
HT-29
HT1080
HepG2
A-549
Panc-1
LNCaP
A-431

⇒ For more details; please view the attached Application Note. In addition; we have received customer feedbacks for the following cell lines:

HCT-116 (human colorectal carcinoma cell line)
Capan-1 (human pancreatic adenocarcinoma cell line)
PC3 (human prostate cancer cell line)
C42B (osteotropic prostate cancer cell line)
NCI-H23 (human lung epithelial adenocarcinoma cells)
IMR-32 (human neuroblast cell line)
A818-6 (human pancreatic ductal adenocarcinoma cell line)
HEK293 (human embryonic kidney cells)
Calu-1 (non-small-cell lung cancer cell line)

hMDM-GMCSF is the abbreviation for human monocyte-derived macrophages. They are polarized [⇒ differentiated with GM-CSF] but non-activated [⇒ (-)] M1 macrophages.
hMDM-MCSF is the abbreviation for human monocyte-derived polarized [⇒ differentiated with M-CSF] but non-activated [⇒ (-)] M2 macrophages.

The macrophages can be seeded into all kinds of TC vessels. After plating; they can be maintained as biologically functional adherent cultures for several weeks.Optionally; user-customizable activation of the cells can be performed. For details; please see attached Application Note; page 2; Fig. 3 and page 5; Tab. 1).

This is not advised. Please seed the freshly isolated CD14-monocytes immediately in the Monocyte Attachment Medium. Adding a culturing step will change the biological characteristics of monocytes very rapidly.

Short protocol:

Thaw the cells (C-12921) for 2 min in a 37°C waterbath. Dilute in 9 ml of complete HPC Expansion Medium XF (+ Cytokine Mix E) and count the cells
Spin down for 10 min at 240xg; aspirate the supernatant; resuspend the pellet at 20;000 cells/ml HPC Expansion Medium XF
Plate in an appropriate suspension culture vessel and incubate the culture for 2-3 days at 37°C and 5% CO₂
Then double the media volume by adding fresh complete medium; e.g.; 4 ml suspension culture + 4 ml fresh medium (= 8 ml)
Incubate the cells for an additional 10-12 days by performing a partial medium change every 2-3 days Example partial medium change: For a culture volume of 8 ml; spin down the cells; aspirate and discard 4 ml of the supernatant; resuspend the cells and add 12 ml of fresh complete medium (= 16 ml).

In combination with the Cytokine Mix E; the HPC Expansion Medium XF typically promotes a 300-1;000 fold expansion of the total cell population. After 2 weeks of expansion about 20-30% of the population express CD34⁺; indicating a 50-200 fold expansion of CD34⁺ progenitor cells.

Yes it is possible to refreeze them.

Yes; Accutase Solution can be defrosted; aliquoted; and then refrozen. Defrosting: Accutase should be defrosted overnight in the refrigerator or placed in a tub of cold tap water. Do not defrost in a 37°C water bath. Stability: Once thawed; it is stable for at least 2 months in the refrigerator if stored promptly after use.

The components of the PromoCell DetachKit may arrive on occasion with a non-uniform color appearance. This phenomenon is known by PromoCell’s Quality Assurance. It is reversible and does not influence the quality of the product.

Secretory granules called Weibel-Palade bodies (WPBs) containing Von Willebrand factor (vWF) are more linked to the formation of a confluent endothelial monolayer. Research shows that vWF expression is dynamic and highly dependent on the cell culture conditions such as confluence and passage number. Therefore; having vWF as a quality control marker for each lot of ECs is not really necessary.

The components of the PromoCell DetachKit may occasionally arrive with a non-uniform color appearance. This phenomenon is known to PromoCell’s Quality Assurance Dept. It is reversible and has no influence on the quality of the product.

We did not plate the cryo-macrophages in 96-well plates. However we heard from other customers that they have successfully used our macrophages in this kind of plate.

You may find all information regarding the activation and the cytokine concentrations in table 1 (page 5) of our Application Note.

No we do not provide data about the cytokine profile of our M1/M2 macrophages after activation and we do not provide any further data beyond the scope of our quality control.

Even non-activated macrophages do release a certain amount of cytokines. Furthermore you would have to be sure that the release of a certain cytokine is a direct consequence of the activation. Therefore we do not think it is possible to have a general negative control for the cytokine release.

The recommended seeding density with 100;000 cells per cm²is needed for a confluent cell layer as the cells do not proliferate. However; you can reduce the seeding density by the factor 3 to 5 and the macrophages are still viable.

We did not test if the macrophages attach on fibronectin-coated glass.

In general; we recommend activating the cells for 24 hours or at least over night for all kind of activations. If the activation is not optimal in your experimental setting; you can increase or decrease the activation time accordingly.

The reason for the higher number of lymphocytes in the macrophage culture is probably due to an insufficient washing step during the purification of the monocyte via adherence. The 3 washing steps in our protocol are essential to receive a monocyte population of over 90%.

Unfortunately we did not test this in our hands and it must be tested by the customer. In fact our medium is completely different from RPMI and therefore we cannot predict if this is working. We only know the successful long-term culture from our system with our media.

M1 / M2 polarization also takes seven days in the PromoCell system but the protocol contains two more days for optional macrophage activation. If you only want non-activated M1 / M2 macrophages; the process is usually completed after 7 days. Nevertheless; PromoCell does not recommend shortening the 10-day protocol because you actually get a plus in viability and cell yield (due to the re-attachment of floating cells) on day 8-10 due to the media change.

At PromoCell; we have not tested macrophage differentiation from PBMC in 96-well plates; but we know from users that it is possible. According to a customer the mononuclear cells differentiate very well in the 96-well format. A plating density of 1 million PBMCs (without prior determination of monocyte content) per well has been shown to be optimal. The working volume in a 96-well plate is usually 100 µl.

No; our Freezing Medium Cryo-SFM (C-29910) is not produced under GMP standard. It is for in vitro research use only and not appoved for diagnostic or therapeutic procedures.

Yes; you can use 4.5% neutral buffered formalin. Paraformaldehyde should work as well.

Osteogenic differentiation of hMSCs with PromoCell MSC Osteogenic Differentiation Medium takes ∼12-14 days (please view the respective Application Note for a detailed protocol). Use fibronectin-coated plates and change the medium every third day. The bone cells tend to detach from the plastic after approximately 2 weeks; when differentiation is complete. Therefore; the tests should be performed promptly and the cells should be maintained in MSC Osteogenic Differentiation Medium until then.

We recommend to use Human Serum AB „off-the-clot”.

Yes, our NHEM.f will also grow in our optimized Melanocyte Growth Medium M3 (C-24310) and can achieve > 15 population doublings.

Yes; the NHEK-GM2 (C-12001; C-12003; C-12005; C-12006) also grow in PromoCell Keratinocyte Growth Medium 3 (C-20021). Using the protocol with the fixed intervals; they grow slightly faster than in Keratinocyte GM2 and proliferate for > 15 PDs. Conversely; NHEK-GM3 (C-12011; C-12013; C-12015; C-12016) also grow in the existing Keratinocyte Growth Medium 2 (C-20011). When using the classical subcultivation protocol (density > 70% - 90%); they grow slightly slower compared to Keratinocyte GM3; but also reach > 15 PDs.

The NHEK (primary human keratinocytes) are isolated in our serum-free Keratinocyte Growth Medium 2 (C-20011) the NHEK GM3 in our improved serum-free and BPE-free Keratinocyte Growth Medium 3 (C-20021). Both NHEK and NHEK GM3 are available from single or from pooled donors isolated from the epidermis of juvenile foreskin or adult skin.

Yes it is possible to aliquot the 5 ml Cytokine Mix E (C-39891) into 5 x 1 ml.

The activation of macrophages as such is complete after 24 hrs. However; to maintain the activation status over a longer period of time (i.e.; several days); fresh activation factors should be added with every medium change.

The basis for the intended use of our products is defined in our Terms & Conditions under the chapter “Use of Goods”.

Our HPAEC and HPASMC are isolated from the proximal pulmonary artery.

You can also use Oil Red O to stain lipid droplets. At PromoCell; we used to use Oil Red O as well; but switched to Sudan III some time ago for organizational reasons.

Light flocculation may be seen upon thawing the supplements containing ECGS/heparin or BPE. This does not affect the activity of our media. Optionally; the precipitate can be removed by centrifugation under sterile conditions. We recommend to thaw the supplements (SupplementMix or SupplementPack) at 15-25°C.

Yes; there are a few differences: - We recommend replacing the MSC Growth Medium XF (C-28019) 3-4 h after seeding; as opposed to 16-24 hours after seeding for most other cell types/growth media. - When MSC Growth Medium XF; MSC Neurogenic Differentiation Medium; MSC Adipogenic Differentiation Medium 2 or MSC Osteogenic Differentiation Medium are used; flasks have to be coated with 10 µg/cm² (human or bovine) fibronectin according to the instruction manual. - We strongly recommend using Accutase (C-41310) for cell detachment instead of Trypsin. If Trypsin is used; contact time should not exceed 2 min.

The supplement should be at room temperature when added to the MSC Adipogenic Basal Medium 2. It may also be beneficial to invert the tube a few times to bring precipitates back into solution. Please note: It is not recommended to filter the basal medium; supplements; or complete medium; as components that induce or promote differentiation may be removed; resulting in a low differentiation rate when using the medium.

According to the product manual Cryo-SFM should be stored at 4-8°C. However since this solution is used to freeze cells in liquid nitrogen we assume that storing Cryo-SFM once at -20°C should not have a negative impact on the product quality. After thawing please store it at 4-8°C as recommended.

The use of antibiotics creates a false sense of security and allows users to develop poor aseptic techniques. This leads to low-level contamination with partially resistant bacteria occurring but being overlooked for a time. This then leads to cells with undetected contamination being cultured for extended periods of time; increasing the risk that contamination will spread throughout the laboratory and eventually antibiotic-resistant strains of bacteria may develop. Mycoplasma infections can also occur more easily; as they are often introduced along with contaminants such as bacteria and fungi. Last but not least; antibiotics are known to have negative effects on the metabolism of eukaryotic cells - more details on this topic can be found in our blog article "Antibiotics in cell culture: friend or enemy".

The PBS buffering enhances the Alizarin Red staining (precipitation of the dye) and makes it more intense. Leave the PBS on the cells after staining/washing and analyze the sample immediately; as the dye may bleed upon prolonged storage without embedding.

PromoCell's Normal Human Epidermal Keratinocytes (NHEK) from juvenile donors are isolated from both the epidermis of the outer and mucosal (i.e. inner) layers of the foreskin. Thus; each vial contains a mixture of mucosal and cutaneous keratinocytes.

Yes; there is a reference (Campuzano et al.; J Immunol. 2020 Jun 15;204(12):3296) where bone marrow-derived macrophages from mouse were detached using our Macrophage Detachment Solution (40 minutes at 4°C).

We guarantee > 500;000 viable cells per vial. To do this; we need to freeze more than 500;000 cells; as we do not yet know the viability after thawing at the time of freezing. For technical/organizational reasons; the initial cell number may also vary from lot to lot; so 2 lots with the same viability may not necessarily contain the same number of cells.

PromoCell is using a pan-cytokeratin antibody rather than a specific type of cytokeratin for the quality control of our keratinocytes.

Yes. The orientation can be triggered by the use or lack thereof of ECM. Without the use of an ECM; the organoids will have a higher outward oriented ratio. You can find a protocol here https://pubmed.ncbi.nlm.nih.gov/30811997/; where cells were first embedded in ECM gel and afterwards ECM was dissociated; and free organoids were re-seeded in suspension without an ECM. * Please note that we have not tested this method in our labs and thus cannot guarantee it.

Possibly. The size of the organoids may depend on the access to nutrients in the gel. For reference; https://pubmed.ncbi.nlm.nih.gov/31173716/ For best results we recommend the following:

Avoid nutrient gradients. We recommend using a small; drop-like BME gel bead and high volumes of medium. Change the medium every day as we’ve observed that less frequent medium changes results in yellow culture medium indicative of a pH shift likely from an increase in cellular metabolism.
For optimal cell distribution; it is important to control the “gelling” or setting of the ECM gel. Therefore; the temperature is critical! BME is liquid in the cold (4°C) and solidifies at 37°C. The gelling must be done in a very short time; otherwise the cells in the BME would sink to the bottom.

ROCKi is known to enhance the proliferation of epithelial cells or keratinocyte progenitors and can improve cell survival via conditional reprogramming and the effect is reversible. It is also known to enhance the seeding efficiency on plastic. This ROCKi conditional reprogramming has also been observed in airway EpCs and cultured organoids:

For our human airway organoids; the use of Y-27632 ROCKi is optional as organoids can still form without it. However; the 3D system has some potential pitfalls like choosing the right plastic or the right ECM. The use of ROCKi may help to keep the system more robust.

It is possible to culture the airway organoids in 96-well U-Bottom plates for suspension cells (e.g.; Greiner Bio-One # 650185). A detailed protocol for the use of 96-well plates can be found in our AppNote. We have not tested 384-well plates or other commercially available plates.

To avoid the spreading of the matrix and to have nice drop-like domes; it is crucial to preheat the plate for 2h at 37°C and to work fast when transferring the gel to the wells. Placing a warming plate under your laminar air flow hood when transferring the gel-cell-mix to the preheated plate may help. From our testing; we found that Nunclon Sphera wells were not ideal for our organoid system as the dome did not stay adhered; but it was able to move on the bottom and the dome became misshaped overtime. However; the altered shape of the dome did not affect the organoids and were successfully cultured for 4 weeks. If movement is not an issue for the user; we recommend that extra caution be taken during media changes by holding the plate at an angle and carefully pipette to remove old medium. Aspirating with vacuum suction can damage or destroy the dome.

Based on negative feedback we have received from customers using bead baths; we strongly discourage the use of bead baths to thaw our cells. It can lead to reduced viability or significantly slower growing cells. If you don’t have a “normal” water bath but only a bead bath in your lab; thaw the vial in a beaker of water in the bead bath. Ensure the water is heated to exactly 37°C using a thermometer placed in the warmed water. Be sure to hold the vial in your hand; and not in a floater; as described in the thawing protocol.

The Cytokine Mix M1 and M2 should not be subjected to further freeze/thaw cycles.

You could probably use our MSC Chondrogenic Differentiation Medium without inducers (C-28014). It is also serum-free and the same as C-28012 just without chondrogenic inducers.

We strongly advise against overnight incubation. The Monocyte Attachment Medium does not contain cytokines/survival factors for the monocytes. If the cells remain in the Monocyte Attachment Medium for longer than 2 hours; they will go into apoptosis and die. The lymphocytes will survive longer. Some of them will attach after such a long time and then cannot be washed off. Therefore; overnight attachment is absolutely inappropriate. However; if time is short; monocyte attachment can be shortened to 1 hour. To do this; it is best to add Monocyte Attachment Medium to the culture vessels in advance and equilibrate it in the incubator. Remove the vessels from the incubator only briefly to add the appropriate amount of concentrated PBMC suspension. Then; even after 1 hour; most of the monocytes are attached.

We have never tested the cultivation of our assay-ready macrophages in MEM alpha + FBS. We cannot predict whether it will work and therefore strongly recommend the use of our M1 Macrophage Generation Medium XF and fibronectin-coated vessels.

Our cells are not manufactured according to GMP guidelines and are intended for in vitro use only. Our EXCiPACT™ GMP certification only applies to the processes related to the media.

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