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
-
How does PromoCell recommend subculturing the chondrocytes (e.g. from T25 into T75 flasks, or into petri dishes)? Does PromoCell recommend a specific type or brand?
We recommend a seeding density for chondrocytes between 10,000 and 20,000 cells/cm². This means that a subconfluent T25-flask with approx. 900,000 cells/T25 flask (36,000 cells/cm² ) may be either split into 3 new T25 or seeded in one T75 flask or in one 100 mm petri dish. We do not recommend a specific type or brand for the culture of HCH.
Related Links and Documents
-
What culture conditions are required for culturing PromoCell Normal Human Cells in the respective PromoCell media?
PromoCell Normal Human Cells should be cultured in the appropriate medium at 37°C and 5% CO2 in a humidified atmosphere.
Please note: If using cell culture flasks w/o filter cap, unscrew the cap by half a turn to allow sufficient ventilation. -
Can you please provide me a protocol for the expansion of CD34+ Progenitor Cells in PromoCell Expansion Medium XF?
Short protocol:
- Thaw the cells (C-12921) for 2 min in a 37°C waterbath. Dilute in 9 ml of complete HPC Expansion Medium XF (+ Cytokine Mix E) and count the cells
- Spin down for 10 min at 240xg, aspirate the supernatant, resuspend the pellet at 20,000 cells/ml HPC Expansion Medium XF
- Plate in an appropriate suspension culture vessel and incubate the culture for 2-3 days at 37°C and 5% CO₂
- Then double the media volume by adding fresh complete medium, e.g., 4 ml suspension culture + 4 ml fresh medium (= 8 ml)
- Incubate the cells for an additional 10-12 days by performing a partial medium change every 2-3 days
Example partial medium change: For a culture volume of 8 ml, spin down the cells, aspirate and discard 4 ml of the supernatant, resuspend the cells and add 12 ml of fresh complete medium (= 16 ml).
In combination with the Cytokine Mix E, the HPC Expansion Medium XF typically promotes a 300-1,000 fold expansion of the total cell population. After 2 weeks of expansion about 20-30% of the population express CD34+, indicating a 50-200 fold expansion of CD34+ progenitor cells.
Related Links and Documents
-
Why does the macrophage differentiation in the PromoCell protocol take 10 days? In the literature or following the feedback from other researchers, it usually only takes 7 days.
M1 / M2 polarization also takes seven days in the PromoCell system but the protocol contains two more days for optional macrophage activation. If you only want non-activated M1 / M2 macrophages, the process is usually completed after 7 days.
Nevertheless, PromoCell does not recommend shortening the 10-day protocol because you actually get a plus in viability and cell yield (due to the re-attachment of floating cells) on day 8-10 due to the media change.Related Links and Documents
-
How can I avoid precipitates when preparing my Mesenchymal Stem Cell Adipogenic Differentiation Medium 2?
The supplement should be at room temperature when added to the MSC Adipogenic Basal Medium 2. It may also be beneficial to invert the tube a few times to bring precipitates back into solution.
Please note: It is not recommended to filter the basal medium, supplements, or complete medium, as components that induce or promote differentiation may be removed, resulting in a low differentiation rate when using the medium.
Related Links and Documents
-
We use Macrophage Generation Media from Promocell to get differentiated macrophages from fresh PBMC. Would it be okay if the attachment step with the Monocyte Attachment Medium is done overnight instead of 1.5 h?
We strongly advise against overnight incubation.
The Monocyte Attachment Medium does not contain cytokines/survival factors for the monocytes. If the cells remain in the Monocyte Attachment Medium for longer than 2 hours, they will go into apoptosis and die. The lymphocytes will survive longer. Some of them will attach after such a long time and then cannot be washed off.
Therefore, overnight attachment is absolutely inappropriate. However, if time is short, monocyte attachment can be shortened to 1 hour. To do this, it is best to add Monocyte Attachment Medium to the culture vessels in advance and equilibrate it in the incubator. Remove the vessels from the incubator only briefly to add the appropriate amount of concentrated PBMC suspension. Then, even after 1 hour, most of the monocytes are attached.
Related Links and Documents
-
Can the media from your cancer media toolbox be used for cells of other, non-human species, e.g., from mice?
Yes, our cancer media (Primary Cancer Culture System/PCCS, 3D Tumorsphere Medium XF, Cancer Cell Line Medium XF) also support the growth of murine tumor cells.
Related Links and Documents
-
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.
Related Links and Documents
-
Does Mesenchymal Stem Cell Growth Medium XF (C-28019) contain Phenol Red?
Yes, our MSC Growth Medium XF contains phenol red. The concentration is confidential.
Related Links and Documents
-
What is the source of the trypsin from the Detach Kit (C-41200/C-41210/C-41220)?
The source is porcine pancreas.
Related Links and Documents
-
Why don’t PromoCell specify in the instructions how long the primary cells should be trypsinized?
The time needed to detach our primary cells depends on many different factors like the cell type, cell density, lot #, trypsin concentration, the efficiency of the washing step before adding the trypsin and the trypsinization temperature.
For most cell types we recommend trypsinization at room temperature and direct observation of detachment under the microscope. This way, you can find out your individual trypsinization time and keep the contact time between cells and trypsin to a minimum. Most cells detach after 2-8 min.Please refer to the instructions in the Manual. For some cell types, trypsinization at 37°C or the use of Accutase or another Detachment Solution is recommended.
Related Links and Documents
-
What is the exact source of PromoCell’s subcutaneous and visceral preadipocytes (HWP)?
Our subcutaneous HWP are isolated from subcutaneous fat of different localizations, e.g. abdomen, breast, or upper arm.
The visceral preadipocytes are isolated from fat surrounding e.g. the pericardium, or from the omentum or mediastinum.The exact localization is specified in the Certificate of Analysis. If you need HWP from a particular localization, please contact our Technical Customer Support prior to placing your order.
Related Links and Documents
-
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
-
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.
Related Links and Documents
-
From what part of the lungs does PromoCell isolate the human pulmonary fibroblasts (HPF)?
Our HPF (C-12360) are isolated from peripheral lung tissue.
Related Links and Documents
-
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
-
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
-
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
-
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).
Related Links and Documents
-
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
-
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.
-
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.
Related Links and Documents
-
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.
Related Links and Documents
-
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.
Related Links and Documents
-
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.
Related Links and Documents
-
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.
Related Links and Documents
-
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.
Related Links and Documents
-
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%.
Related Links and Documents
-
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.
Related Links and Documents
-
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.
Related Links and Documents
-
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
-
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
Related Links and Documents
-
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.
-
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.
Related Links and Documents
-
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.
Related Links and Documents
-
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.
Related Links and Documents
-
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.
-
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.
Related Links and Documents
-
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.
Related Links and Documents
-
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.
Related Links and Documents
-
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
-
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.
Related Links and Documents
-
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
-
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.
Related Links and Documents
-
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
-
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
-
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.
Related Links and Documents
-
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.
Related Links and Documents
-
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.
Related Links and Documents
-
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
Choose your Region
Please choose your region for an optimized website experience. So we can provide you with the most useful information for your country.
- North America
- Europe
- Asia