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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Can I use a serum-free medium as negative control in the chondrogenic MSC differentiation? I would like to replace the recommended DMEM+10% FCS.
You could probably use our MSC Chondrogenic Differentiation Medium without inducers (C-28014). It is also serum-free and the same as C-28012, just without chondrogenic inducers.
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If I isolate tumor cells from my PDX animal model using your PCCS, will the animal cells survive?
In our Primary Cancer Culture System (PCCS, C-28081), only the malignant tumor cells can survive. Therefore, benign animal cells (e.g., fibroblasts) will be depleted and the malignant human tumor cells will survive.
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Is there is a difference in the percentage of differentiated DCs depending on whether we start with cryopreserved or fresh isolated monocytes?
There is no significant difference in the percentage of cells that will differentiate into Dendritic Cells. But when using cryopreserved cells, the initial cell loss will be higher compared to when fresh cells are used. i.e. the final number of differentiated cells that can be expected will be higher with fresh cells as a starting material due to lower cell death rate.
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Can PromoCell release the species the following cytokines were produced in: basic Fibroblast Growth Factor and Epidermial Growth Factor included in the Endothelial Cell Growth Media supplements?
Both cytokines are produced in E.coli.
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What is the differentiation ratio that can be achieved with PromoCell’s subcutaneous HWP?
Most of our subcutaneous preadipocyte lots achieve > 80-90% differentiation when differentiation is induced at P2 (directly after thawing).
We generally recommend using cells for differentiation tests that haven’t undergone more than 4-5 doublings (a maximum of 1 passage after thawing), as the differentiation ratio will decline with the age of the cells.Related Links and Documents
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The HDMEC I purchased from PromoCell seem to contain 2 different cell morphologies. How is this possible? What’s the purity of HDMEC cultures guaranteed by PromoCell?
Our HDMEC are isolated from the dermis of juvenile foreskin or adult skin. The purity is > 95%.
Since the dermis contains blood and lymphatic capillaries, HDMEC cultures comprise blood and lymphatic microvascular endothelial cells that have differing morphologies. Both cell types have a common origin and can be identified by several markers. The ratio of lymphatic and blood derived endothelial cells can vary from lot to lot and is not determined at PromoCell.Related Links and Documents
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What is the basis formula for PromoCell’s Osteoblast Basal Medium (C-27010)?
PromoCell’s Osteoblast Basal Medium is an optimized media formulation developed for human osteoblast culture. The exact composition is proprietary.
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Can I expand the human Mesenchymal Stem Cells prior to differentiating them?
These cells are frozen at the end of 2nd culture. Thawing and seeding results in passage 2 (3rd culture). We recommend that they be used for differentiation experiments not later than passage 5.
The differentiation potential of hMSC in vitro is reduced with ongoing population doublings, meaning the earlier differentiation is induced, the higher the differentiation rates.Related Links and Documents
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What is the lead time when ordering customized media from PromoCell?
The lead time is usually 4-8 weeks.
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Where in the lung are the HPMEC harvested from? Are the cells from arterioles or capillaries?
Our human pulmonary microvascular endothelial cells (C-12281) are sourced from the lung parenchyma with all large vessels being removed beforehand. Therefore, most of the HPMEC originate from capillaries.
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Do the PromoCell HCMEC represent the endocardial cells, i.e. those lining the ventricles? If so, why are they referred to as microvascular endothelial cells?
Our HCMEC (Human Cardiac Microvascular Endothelial Cells) are not endocardial cells. They are isolated from the capillaries in the heart muscle. Therefore, they are in fact microvascular.
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Can I use PBS instead of HepesBSS to wash the cells before trypsinization?
The PromoCell trypsin 0.04% / EDTA 0.03% solution and the PromoCell TNS solution are both based on HepesBSS. Therefore, it is best to also use HepesBSS to wash the cells prior to trypsinization.
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What plating density should I use for PromoCell Normal Human Cells?
The recommended plating density after thawing/subculture may vary depending on the cell type. Please refer to the data sheet for your cells under “Specifications”.
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How can I successfully isolate HUVEC from umbilical cords without using antibiotics?
At PromoCell, we get the umbilical cords from our tissue suppliers with no addition of buffers or media. This method prevents the microorganisms from being washed into the blood vessels. Before we start the cell preparation, the umbilical cord is also cut on both ends with a sterile scalpel to provide sterile intersections in addition to the sterile lumen.
This method allows us to isolate sterile endothelial cells from umbilical vein and to plate them in antibiotics-free culture media.Related Links and Documents
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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.
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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.
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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
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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.
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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.
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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.
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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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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.
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What is the source of the trypsin from the Detach Kit (C-41200/C-41210/C-41220)?
The source is porcine pancreas.
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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.
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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.
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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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