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
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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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What is the concentration of ECGS contained in Endothelial Cell Growth Media/Preadipocyte Growth Media?
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.
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From what part of the lungs does PromoCell isolate the human pulmonary fibroblasts (HPF)?
Our HPF (C-12360) are isolated from peripheral lung tissue.
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What is the difference between juvenile (C-12210) and adult HDMEC (C-12212)? Which ones should I use for my experiments?
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.Related Links and Documents
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Is it necessary to use the PromoCell DetachKit when subculturing PromoCell Normal Human Cells?
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.Related Links and Documents
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I cannot find the Certificate of Analysis (CoA) pertaining to my cells. Can you please send me a copy?
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.Related Links and Documents
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What is the approximate cell density of HFDPC (C-12071) at subconfluence?
Typical cell densities are between 32,000 – 40,000 cells/cm² (approx. 800,000 – 1 million cells per T25-flask).
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Are the HPAEC harvested directly from the pulmonary artery or are these microvascular endothelial cells?
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.Related Links and Documents
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How can I prevent fibroblast contamination in epithelial cell preparations?
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.
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Is it possible to refreeze the hCD34 progenitor cells after having amplifed them in PromoCell Hemaotopoietic Progenitor Cell Expansion Medium XF?
Yes, it is possible to refreeze them.
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Is it possible to differentiate M1 macrophages from PBMC in 96-well plates?
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.
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I have accidently stored the Cryo-SFM at -20°C. Can the product still be used in this case?
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.
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Can PromoCell M1 macrophages be cultured after thawing in MEM alpha containing 10% FBS and GM-CSF?
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.
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Is PCCS the best media for isolation of cancer stem cells from fresh tumor tissue?
Yes, it is. The Primary Cancer Culture System (C-28081) is very selective for cancer stem cells – any other cells will be eliminated after a short time.
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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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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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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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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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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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How should I best freeze normal human cells?
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
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Where can I find the composition of the supplements?
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.
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From which organism originate the recombinant cytokines in Cytokine Mix E (C-39890)?
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.
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Are the PromoCell chondrocytes from orthopedic joint replacement surgery or other types of surgery?
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.
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Will RNAlater solution denature the proteins in the cell pellets?
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.
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Should I store the cryopreserved cells in the liquid phase or gas phase of liquid nitrogen?
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.
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How long is it before the osteoblasts produce mineralized nodules?
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.
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What calcium concentration is needed to induce differentiation in cultured keratinocytes?
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.
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Will precoating of dishes have any adverse impact on the HUVEC?
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.
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Can I defrost Accutase Solution, prepare aliquots and 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.Related Links and Documents
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Is PromoCell’s Cryo-SFM produced under GMP standard?
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.
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What are the disadvantages of the prophylactic use of antibiotics in cell culture?
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”.Related Links and Documents
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Are your cells isolated under GMP?
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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What do I need to consider when culturing melanocytes in your Melanocyte Growth Medium M3?
Our Melanocyte Growth Medium M3 allows the serum-, BPE- and PMA-free cultivation of Normal Human Epidermal Melanocytes (NHEM) without additional coating of the TC plastic.To maintain the cells in a robust adherent pro-proliferative phase, we highly recommend the passaging of cells at 70-90 % confluency.
It is known from the literature that high cell densities of NHEM can promote the growth of 3D spheroids. Therefore, too high confluencies should be avoided.Related Links and Documents
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What is the number of primary cells per vial frozen at PromoCell? How can I calculate the number of viable cells and how should I calculate the optimal plating density?
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).Related Links and Documents
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I am having problems isolating RNA from peripheral blood MNC pellets. Is there any reason you can think of why this would be?
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.
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What is the exact localization of PromoCell’s HRCEpC? Are the cells isolated from proximal or distal tubuli?
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.
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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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When subculturing PromoCell’s proliferating HUVEC (T25 flask; C-12250), which TC flask should I use?
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).Related Links and Documents
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At what passage should I induce the differentiation of the SkMC?
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.
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Where can I find the composition of your basal media?
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.
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What chondrocyte structures are stained using Alcian Blue? Can I use this staining method for chondrocytes in monolayer cultures?
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.Related Links and Documents
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Does PromoCell recommend growing their osteoblasts on type I collagen?
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.
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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 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.
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What experience do I need to grow PromoCell Normal Human Cells, Adult Stem and Blood Cells?
You need to have experience working under sterile conditions and under a laminar flow hood. It is of advantage to have experience with other cell types and/or cell lines. If you are a beginner in cell culture and would like to establish a cell culture lab, we will assist you in working with PromoCell Normal Human Cells.
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How many osteoblasts will be in the flask when I purchase a proliferating culture (C-12760)?
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).
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Typically once seeded, how long does it take to grow the chondrocytes to subconfluence? How many doublings will they undergo per passage?
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.
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Is it necessary to cultivate Nasal Epithelial Cells on coated culture flasks?
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.
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The components of my DetachKit show different colors. Is this normal or does it affect their quality?
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.
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I am performing MSC chondrogenic, adipogenic, and osteogenic differentiation and am staining the cells. The protocol says to use Saccomanno Fixation Solution. Is there an alternative method to fix the cells because I don’t have Saccomanno Fixation Solution?
Yes, you can use 4.5% neutral buffered formalin. Paraformaldehyde should work as well.
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Why is PBS added in the last step of the Alizarin Red staining protocol (“Osteogenic differentiation and analysis of MSC”)? Should the PBS be aspirated before analysis?
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.Related Links and Documents
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What is the shelf life of primary cells cryopreserved in liquid nitrogen?
PromoCell cells are frozen down in the gas phase of liquid nitrogen (computer-controlled freezing machine) and then stored in the liquid phase of LN2.
The cryopreservation in LN2 is an acknowledged method for long-term storage of primary cells and stem cells. When stored in liquid nitrogen, the cells can be maintained for a period of > 10 years without affecting viability.
For example, Kumar et al. showed that adipose-derived stem cells stored in LN2 for about 12 years still retained their regenerative potential, stem cell property, viability as well as differentiation ability.
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Could you please let me know whether you have used the baculovirus expression system to produce components of your media?
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. -
Do I need precoated flasks when growing my MSC in PromoCell Mesenchymal Stem Cell Growth Medium XF (C-28019)?
Our MSC Growth Medium XF provides a xeno-free culture system for human MSCs. It contains all growth factors and supplements except attachment- and spreading factors.
Therefore, culture vessels to be used with Mesenchymal Stem Cell Growth Medium XF must be precoated either with 1 μg/cm2 human fibronectin or 0.5 μg/cm2 human vitronectin according to the instruction manual of the manufacturer.
Alternatively, bovine fibronectin may be used.Related Links and Documents
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Is it normal that CD34+ 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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How should I handle PromoCell’s proliferating cells after arrival?
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.Related Links and Documents
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I have a question related to PromoCell’s CD34+ Progenitor Cells (C-12921). Are they suspension cells or do they partially attach?
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.Related Links and Documents
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Can rat or mouse SkMCs be cultured using the Skeletal Muscle Cell Growth Medium (C-23060)?
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.Related Links and Documents
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When should I order the Growth Medium Kit instead of the Medium “ready-to-use”?
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.Related Links and Documents
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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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Can PromoCell normal human cells be grown in standard media like DMEM or RPMI1640 supplemented with 10% FBS?
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.
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Can I isolate RNA and/or proteins from the cell pellets?
PromoCell cell pellets (C-14**) are an easily accessible source of DNA, RNA, and proteins.
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How many population doublings do PromoCell guarantee for the Normal Human Cells?
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”.
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Can I first expand the osteoblasts and then perform my experiments?
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.
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Does the Chondrocyte SupplementMix (C-39635) contain any growth factors?
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.
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Where does PromoCell source the bronchial tissue for HBEpC preparation? Are the smoking habits of the donors known?
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.Related Links and Documents
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Is vWF a great marker for Endothelial Cells?
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.Related Links and Documents
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I would like to differentiate hMSC into bone cells and further maintain the cells in culture after differentiation. Which medium should I use?
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.
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Are your NHEK from the epidermis of the juvenile foreskin mucosal or cutaneous keratinocytes or a mixed population?
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.
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I would like to know if, in addition to HBEpC, the other airway epithelial cells are also compatible with PromoCell ALI medium.
- For our HNEpC and HTEpC, please refer to the instructions in the AppNote describing the differentiation of these cell types using our ALI medium.
- We do not provide instructions for HSAEpC because our medium is not suitable for differentiation of this cell type at the air-liquid interface.
- For our HBEpC we have special ALI prescreened lots in stock that were successfully tested for barrier function in our QC. Please contact our technical customer support for more information.
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For how long can the monocyte-derived DCs be maintained in culture after the differentiation is completed (day 7/8 of the protocol)?
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".
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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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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 are the different types of multiwell plates that can be used in fluorescence, luminescence and colorimetric detection? Which type should I use for which application?
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
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Which of the PromoCell DetachKits, C-41200 or C-41202 should I use to trypsinize the HNEpC?
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.
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From what muscles does PromoCell isolate the SkMC?
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.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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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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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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Can the cell pellets be used to establish a growing culture?
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.
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How many passages can I perform with PromoCell Normal Human Cells?
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”. -
Does the Osteoblast Growth Medium support osteoblast proliferation or differentiation? Does the medium contain any recombinant growth factors?
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).
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Is it feasible to store the melanocytes for a second time in liquid nitrogen after subculture, similar to cell lines?
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 our Cryo-SFM (C-29910). It is also recommended to freeze the cells at an early passage.
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What is the exact localization of PromoCell’s HNEpC (C-12620)?
Our Nasal Epithelial Cells are isolated from nasal septum or adenoids.
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Are endothelial cells α-SMA-negative under all circumstances?
Scientific findings from different groups, as well as our own results indicate that the presence or absence of α-SMA is not a valid indicator for the composition of a given cell population. Research findings and our inhouse data suggest that endothelial cells are not α-SMA-negative under all circumstances. α-SMA negativity is not an intrinsic property of endothelial cells but can vary depending on extrinsic influences.
Lot-specific values for α-SMA in our Certificates of Analysis (CoAs) are therefore not considered meaningful.
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Which human AB serum does PromoCell recommend to generate M0 macrophages?
We recommend to use Human Serum AB „off-the-clot”.
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Can your Macrophage Detachment Solution also be used for bone marrow-derived macrophages from mice?
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).
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Can I use frozen PBMCs instead of CD14+ monocytes to generate dendritic cells with PromoCell DC Generation Medium?
PBMCs (= peripheral blood mononuclear cells) consist mainly of lymphocytes and monocytes. Cryopreservation causes the CD14+ monocytes to significantly lose their ability to attach to TC plastic. Therefore, the use of frozen PBMCs as starting material for DC generation is not possible because the purification step with Monocyte Attachment Medium will not work.
You can either start with cryopreserved CD14+ monocytes (C-12909) or use freshly isolated mononuclear cells or fresh CD14+ monocytes.
Please refer to the Product Manual of C-28050 or our Application Note [Generation of monocyte-derived Dendritic Cells] for the appropriate protocol.Related Links and Documents
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From which part of the leg does PromoCell isolate the HSaVEC?
The Vena saphena section that we use for HSaVEC isolation originates from the thigh.
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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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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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What’s the difference between visceral and subcutaneous HWP? When should I use which cell type?
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.Related Links and Documents
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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 ac-LDL uptake and are delivered in P2. The recommended culture medium is Endothelial Cell Growth Medium MV2 (C-22022). -
How should I use accutase for cell detachment?
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.
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What is the difference between DetachKit (C-41200) and DetachKit-2 (C-41202)?
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.
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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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Do I have to use coated tissue culture vessels for culturing PromoCell Normal Human Cells?
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- or Vitronectin-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- or Vitronectin-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.
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At what passage do PromoCell test the cell type-specific markers?
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. -
What is the definition of “adult stem cells”?
“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).
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What is the time frame for the differentiation of HWP? Can PromoCell provide a differentiation protocol?
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-27437) 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.
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The melanocytes from Promocell are primary cells, so how many passages can they go before losing activity?
PromoCell guarantee 15 population doublings for their NHEM. In terms of passages this corresponds to approx. 4-6 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.
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