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 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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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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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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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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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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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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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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