Technical Library
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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.
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".
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).
- Right coronary artery
- Left main coronary artery
- Circumflex coronary artery and
- Left anterior descending coronary artery.
- 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 FalconTM.
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).
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.
- 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.
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.
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.
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.
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
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.
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.
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).
These cells are frozen at the end of 2nd culture. Thawing and seeding results in passage 2 (3rd 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
- 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.
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.
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%.
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 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.
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.
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.
- 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.
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.
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.
- 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.
- 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.
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.
- 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.
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.
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).
- 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).
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.
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.
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.
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 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.
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.
- U-87 MG
- MCF-7
- MDA-MB-231
- HT-29
- HT1080
- HepG2
- A-549
- Panc-1
- LNCaP
- A-431
- 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.
- 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).
Yes, our NHEM.f will also grow in our optimized Melanocyte Growth Medium M3 (C-24310) and can achieve > 15 population doublings.
- 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.