Have a question about one of our products? Check out our technical library for recently asked questions from other scientists around the world.
Technical Library
Recent Entries in Technical Library
277 Entries on 6 Pages
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What antibiotics concentration should I add to the PromoCell media?
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 BPlease note: Addition of antibiotics can reduce the growth rate of the cells.
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Are the Renal Epithelial Cells isolated from proximal or distal tubuli?
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.Related Links and Documents
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How does PromoCell characterize their kidney cells (HREpC; HRCEpC) ?
Our kidney cells are characterized by their epithelial cell morphology and by cytokeratin expression using a pan-cytokeratin antibody.
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What is the difference between PromoCell’s HREpC and HRCEpC?
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.
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What could be the reason for a lower yield when using M2 Macrophage Generation Medium compared to M1 Macrophage Generation Medium for a culture started with the same monocytes?
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.
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What is the differentiation ratio that can be achieved with PromoCell’s subcutaneous HWP?
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.Related Links and Documents
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After detaching the macrophages for flow cytometric analysis, I noticed that the washing and centrifugation steps take a long time. Can I shorten the spin time to 10 minutes at 350 x g?
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.
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Is it normal that CD34+ or CD133+ progenitor cells attach to tissue culture plastic after a week-long culture in HPC Expansion Medium XF (C-28021)?
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.
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At what passage are PromoCell Human Blood Cells upon arrival?
PromoCell Blood and Blood Progenitor Cells are cryopreserved directly after isolation (= P0). They haven’t been in culture before freezing.
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Will the hematopoietic progenitor cells proliferate in Hematopoietic Progenitor Cell Expansion Medium XF (C-28021) without Cytokine Mix E (C-39890)?
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–.Related Links and Documents
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What markers does PromoCell test to characterize their Adult Human Stem and Blood 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”).
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What happens if I grow CD34+ or CD133+ progenitors in Hematopoietic Progenitor Cell Expansion Medium XF (C-28021) without Cytokine Mix E (C-39890)?
The cells will die. Hematopoietic Progenitor Cell Expansion Medium XF always must be supplemented with Cytokine Mix E or an appropriate cocktail of cytokines.
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What is the difference between DC Generation Medium XF (C-28052) and DC Base Medium XF (C-28054)?
- 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.
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What are the advantages of using PromoCell DC Generation Medium XF for dendritic cell generation?
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.
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I would like to know if the Lymphocyte Separation Medium (C-44010) is endotoxin tested.
Yes, our Lymphocyte Separation Medium 1077 is endotoxin-tested. The specification for the product is < 10EU/ml.
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In the past, we used MSC Growth Medium (C-28010) in our lab. Can I use MSC Growth Medium 2 (C-28009) instead? What is the difference?
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.Related Links and Documents
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How do I best isolate RNA from PromoCell cell pellets (C-14**)?
There are two options for isolating RNA from cells stored in RNAlater Solution:
1) The solution is removed from the cells prior to extraction. Because of the density of RNAlater Solution, greater centrifugal forces are required to spin down the cells. We recommend to transfer the content of the vial into a 15 ml conical tube, to add an equal volume of PBS and to spin down the cells at 5,000 x g for 10 minutes at 4°C.
2) Alternatively, cells in RNAlater Solution can be used directly for RNA extraction. Because of the greater volume (200 µl), this method generally requires additional lysis solution. -
Which plates does PromoCell recommend as 96-well U bottom suspension plates for chondrogenic differentiation of Human Mesenchymal Stem Cells?
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.
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Does the differentiation capacity of Human Mesenchymal Stem Cells change over time? When should I best induce differentiation?
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.
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Can I detect the differentiation of Mesenchymal Stem Cells by their morphology, without doing any cell staining?
- 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.
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How do mesenchymal stem cells from different tissues differ in terms of their biological function?
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.
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Can I expand the human Mesenchymal Stem Cells prior to differentiating them?
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.Related Links and Documents
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What is the expected differentiation rate when using PromoCell Mesenchymal Stem Cell Osteogenic Differentiation Medium with hMSC-BM?
The differentiation rate into the osteogenic lineage is 70-100%.
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Does PromoCell offer a special 3-D system to block de-differentiation or does PromoCell have special culture conditions that stabilize the chondrocyte phenotype?
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.Related Links and Documents
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When should I use DetachKit, when DetachKit-2?
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).Related Links and Documents
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At what passage are PromoCell Mesenchymal Stem Cells / Pericytes upon arrival?
Our Human Mesenchymal Stem Cells and Human Pericytes are cryopreserved at the end of secondary culture (P1). After thawing, they are in P2.
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How big is the portion of MSC that can be differentiated into neuronal lineages?
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.
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How does PromoCell characterize their MSC-AT (C-12977)?
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.
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After differentiation of human MSC into mature adipocytes – what medium do I have to use to culture those cells?
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).
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How long can I keep neuronally differentiated MSC in culture?
Changing the medium every 2-3 days, the neuronally differentiated MSC can be kept in culture for up to 2 weeks.
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I would like to know how homogenous the MSC population is and if all cells become osteoblasts when grown in MSC Osteogenic Differentiation Medium (C-28013).
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.
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What population doubling times can I expect when growing PromoCell’s Mesenchymal Stem Cells? How long does it take from seeding to subculture?
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.Related Links and Documents
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The Certificate of Analysis of hMNC contains the percentages of the main cell types (lymphocytes, monocytes, …). How does PromoCell determine that these are monocytes?
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.
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How long does it take to differentiate monocytes into mature DCs using PromoCell DC Generation Medium/DC Generation Medium XF?
It takes 7-8 days to generate fully mature myeloid dendritic cells.
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Which DC medium does PromoCell recommend for freshly isolated MNC or monocytes, which one for cryopreserved monocytes?
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.Related Links and Documents
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Does PromoCell recommend a specific plasticware to use for the generation of dendritic cells from peripheral blood monocytes?
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.
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Is there is a difference in the percentage of differentiated DCs depending on whether we start with cryopreserved or fresh isolated monocytes?
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.
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We use Macrophage Generation Media from PromoCell to get differentiated macrophages from peripheral blood monocytes. The monocytes are isolated by culturing PBMCs in Monocyte Attachment Media (also from PromoCell) for 1.5 h. Would it be ok if the attachment step is done overnight, since we receive the blood later in the day which makes it difficult to process on the same day?
It is not recommended to leave the blood cells in the Monocyte Attachment Medium for longer than 1.5-2 hrs. The medium was developed for (short-term) attachment of the monocytes and does not provide nutrients for a longer time period. Leaving the cells in Monocyte Attachment Medium for a longer time or even overnight will induce apoptosis and lead to the loss of the cells.
If necessary, you can reduce the incubation time to 1 hr. In this case, it is advisable to equilibrate the media in the incubator before so that you can immediately and directly add the appropriate amount of PBMC suspension.Related Links and Documents
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What are the advantages of monocyte enrichment by adherence selection using your Monocyte Attachment Media (C-28051)?
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.
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How can I test whether my cells are infected with mycoplasma?
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.
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Can mycoplasma contamination be observed with the naked eye?
No, mycoplasma can only be observed through electron microscopy. For highly sensitive detection of mycoplasma contamination, we recommend the use of PCR-based mycoplasma tests.
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How does PromoCell determine the phototype of skin tissue donors?
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.
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Is it possible to carry out the macrophage differentiation in either a 96- or 384-well plate, so long as the volumes are adjusted accordingly? If not, is it possible for cells to be detached and re-plated in the desired culture plate?
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.).Related Links and Documents
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Do you know in what media the monocyte-derived DCs can be maintained in culture after the differentiation is completed?
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.
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Why is Human Serum Albumin (HSA) added to the PBS wash after detachment of macrophages with Macrophage Detachment Solution?
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.
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Which medium do PromoCell recommend to use on HUVEC – Endothelial Cell Growth Medium or Endothelial Cell Growth Medium 2?
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.
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What is the exact localization of Human Dermal Lymphatic Endothelial Cells (HDLEC)? What medium should I use to culture them?
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).Related Links and Documents
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I am planning to use Endothelial Cell Growth Medium (C-22010) to grow my HUVEC. Do I have to supplement the medium with additional factors like e.g. FCS?
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.Related Links and Documents
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What’s the difference between PromoCell HUVEC single donor (C-12200) and HUVEC-pooled (C-12203)?
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).Related Links and Documents
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What is the source of the heparin that comes with the Endothelial Cell Growth Media (C-22010/C-22011/C-22020)?
The source of our heparin is ex porcine mucosa.
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