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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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 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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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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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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Are there any differences in the cultivation protocol when cultivating MSCs compared to other cell types?
Yes, there are a few differences:
– We recommend replacing the MSC Growth Medium XF (C-28019) 3-4 h after seeding, as opposed to 16-24 hours after seeding for most other cell types/growth media.
– When MSC Growth Medium XF, MSC Neurogenic Differentiation Medium, MSC Adipogenic Differentiation Medium 2 or MSC Osteogenic Differentiation Medium are used, flasks have to be coated with 10 µg/cm² human (C-43060) or bovine (C-43050) fibronectin according to the instruction manual.
– We strongly recommend using Accutase (C-41310) for cell detachment instead of Trypsin. If Trypsin is used, contact time should not exceed 2 min.Related Links and Documents
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After thawing the supplements, I see some precipitation. Is this normal?
Light flocculation may be seen upon thawing the supplements containing ECGS/heparin or BPE. This does not affect the activity of our media.
Optionally, the precipitate can be removed by centrifugation under sterile conditions.We recommend to thaw the supplements (SupplementMix or SupplementPack) at 15-25°C.
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We are working with PromoCell MSCs and MSC Adipogenic Differentiation Medium. Do you have any information on a comparison between Sudan III and Oil Red O and if Sudan III is more efficient at labeling lipid vesicles?
You can also use Oil Red O to stain lipid droplets. At PromoCell, we used to use Oil Red O as well, but switched to Sudan III some time ago for organizational reasons.
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Are PromoCell HPAEC and HPASMC obtained from the proximal or distal part of the pulmonary artery?
Our HPAEC and HPASMC are isolated from the proximal pulmonary artery.
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Why are PromoCell products “for in vitro research use only”?
The basis for the intended use of our products is defined in our Terms & Conditions under the chapter “Use of Goods”.
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How long do I need to activate my macrophages? Can I stop adding activation factors after 24 hours?
The activation of macrophages as such is complete after 24 hrs. However, to maintain the activation status over a longer period of time (i.e., several days), fresh activation factors should be added with every medium change.
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Can the Cytokine Mix E for the Hematopoietic Progenitor Expansion Medium XF (C-39891; 5 ml) be safely aliquoted?
Yes, it is possible to aliquot the 5 ml Cytokine Mix E (C-39891) into 5 x 1 ml.
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What is the difference between PromoCell NHEK and NHEK GM3?
The NHEK (primary human keratinocytes) are isolated in our serum-free Keratinocyte Growth Medium 2 (C-20011), the NHEK GM3 in our improved serum-free and BPE-free Keratinocyte Growth Medium 3 (C-20021). Both, NHEK and NHEK GM3 are available from single or from pooled donors isolated from the epidermis of juvenile foreskin or adult skin.
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Will my NHEK (isolated in PromoCell Keratinocyte Growth Medium 2) grow in PromoCell Keratinocyte Growth Medium 3?
Yes, the NHEK-GM2 (C-12001, C-12003, C-12005, C-12006) also grow in PromoCell Keratinocyte Growth Medium 3 (C-20021). Using the protocol with the fixed intervals, they grow slightly faster than in Keratinocyte GM2 and proliferate for > 15 PDs.
Conversely, NHEK-GM3 (C-12011, C-12013, C-12015, C-12016) also grow in the existing Keratinocyte Growth Medium 2 (C-20011). When using the classical subcultivation protocol (density > 70% – 90%), they grow slightly slower compared to Keratinocyte GM3, but also reach > 15 PDs.Related Links and Documents
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Will the NHEM.f (C-12400) and NHEM.f M2 (C-12402) also grow in PromoCell Melanocyte Growth Medium M3?
Yes, NHEM.f and NHEM.f M2 also grow in our optimized Melanocyte Growth Medium M3 (C24310) and can achieve > 15 population doublings.
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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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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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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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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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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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Can the medium for M2 macrophages be switched into RPMI or RPMI + M-CSF after differentiation?
Unfortunately, we did not test this in our hands, and it must be tested by the customer. In fact, our medium is completely different from RPMI and therefore we cannot predict if this is working. We only know the successful long-term culture from our system with our media.
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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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I differentiated M1 macrophages from self-sourced PBMCs following your differentiating protocol. In the final cell culture, I can see small cells attached to the macrophages which are CD3+ (T-cells). What is the reason for the T-cell contamination after the differentiation?
The reason for the higher number of lymphocytes in the macrophage culture is probably due to an insufficient washing step during the purification of the monocyte via adherence. The 3 washing steps in our protocol are essential to receive a monocyte population of over 90%.
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How long do I have to activate the cryopreserved macrophages to measure cytokine release?
In general, we recommend activating the cells for 24 hours or at least over night for all kind of activations. If the activation is not optimal in your experimental setting, you can increase or decrease the activation time accordingly.
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I would like to analyze the macrophages by microscope and I would therefore have to plate the cells on fibronectin-coated glass. Did you test if the macrophages grow on fibronectin-coated glass?
We did not test if the macrophages attach on fibronectin-coated glass.
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Following the recommended seeding density for your M1 macrophages (100,000 cells per cm2), I would need too many cells to have a complete 96- or 384-well plate. This is too expensive for me, can I reduce the number of cells per cm2?
The recommended seeding density with 100,000 cells per cm2 is needed for a confluent cell layer as the cells do not proliferate.
However, you can reduce the seeding density by the factor 3 to 5 and the macrophages are still viable.Related Links and Documents
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Do you recommend a certain negative control for the activated macrophages to compare the cytokine release with?
Even non-activated macrophages do release a certain amount of cytokines. Furthermore, you would have to be sure that the release of a certain cytokine is a direct consequence of the activation. Therefore, we do not think it is possible to have a general negative control for the cytokine release.
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Does PromoCell have any data regarding the cytokine profile and different markers of the M1/M2 macrophages after activation?
No, we do not provide data about the cytokine profile of our M1/M2 macrophages after activation and we do not provide any further data beyond the scope of our quality control.
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Which cytokine concentrations does PromoCell recommend for activating M1/M2 macrophages?
You may find all information regarding the activation and the cytokine concentrations in table 1 (page 5) of our Application Note.
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Did PromoCell try plating the cryo-macrophages in 96-well plates?
We did not plate the cryo-macrophages in 96-well plates. However, we heard from other customers that they have successfully used our macrophages in this kind of plate.
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I received 3 DetachKits yesterday. Some of the tubes have different colors, although they all come from the same batch.
The components of the PromoCell DetachKit may occasionally arrive with a non-uniform color appearance. This phenomenon is known to PromoCell’s Quality Assurance Dept.
It is reversible and has no influence on the quality of the product.Related Links and Documents
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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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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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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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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 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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Why are the cryopreserved macrophages abbreviated as hMDM-GMCSF(-) and hMDM-MCSF(-), respectively? What does the (-) stand for?
- hMDM-GMCSF is the abbreviation for human monocyte-derived macrophages. They are polarized [⇒ differentiated with GM-CSF] but non-activated [⇒ (-)] M1 macrophages.
- hMDM-MCSF is the abbreviation for human monocyte-derived polarized [⇒ differentiated with M-CSF] but non-activated [⇒ (-)] M2 macrophages.
The macrophages can be seeded into all kinds of TC vessels. After plating, they can be maintained as biologically functional adherent cultures for several weeks.Optionally, user-customizable activation of the cells can be performed. For details, please see attached Application Note, page 2, Fig. 3 and page 5, Tab. 1).
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If I isolate fresh CD14-monocytes for M1/M2 macrophage generation, can I culture them for a period of time before differentiation?
This is not advised. Please seed the freshly isolated CD14-monocytes immediately in the Monocyte Attachment Medium. Adding a culturing step will change the biological characteristics of monocytes very rapidly.
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Can you provide a list of cell lines that have been successfully used for tumorsphere formation with the PromoCell 3D Tumorsphere Medium XF?
The following cell lines have been tested at PromoCell to form tumorspheres in 3D Tumorsphere Medium XF (C-28070):
- U-87 MG
- MCF-7
- MDA-MB-231
- HT-29
- HT1080
- HepG2
- A-549
- Panc-1
- LNCaP
- A-431
⇒ For more details, please view the attached Application Note.
In addition, we have received customer feedbacks for the following cell lines:- HCT-116 (human colorectal carcinoma cell line)
- Capan-1 (human pancreatic adenocarcinoma cell line)
- PC3 (human prostate cancer cell line)
- C42B (osteotropic prostate cancer cell line)
- NCI-H23 (human lung epithelial adenocarcinoma cells)
- IMR-32 (human neuroblast cell line)
- A818-6 (human pancreatic ductal adenocarcinoma cell line)
- HEK293 (human embryonic kidney cells)
- Calu-1 (non-small-cell lung cancer cell line)
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Can PromoCell MSC Growth Medium 2 (C-28009) be used for Mesenchymal Stem Cells from rat?
Yes, we have received a customer feedback that our MSC Growth Medium 2 also works for rat MSCs.
The rat cells grow nicely in this medium and have a good viability.Related Links and Documents
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Which medium does PromoCell recommend for endothelial cell transfection ?
There are several factors that can influence successful transfections, e.g. viability and density of the cells, choice of the transfection reagent, quality and type of the transfected molecules (plasmids, siRNA, oligonucleotides), as well as the culture medium and supplements used.
When using PromoCell Endothelial Cell Growth Media for cell transfection, please follow the instructions below:Heparin, which is included in our Endothelial Cell Growth Medium (C-22010/C-22110), Medium 2 (C-22011/C-22111) and Medium MV (C-22020/C-22120) may reduce the transfection efficiency. We therefore recommend to use our heparin-free Endothelial Cell Growth Medium MV2 (C-22022/C-22121). Alternatively, you may use Endothelial Cell Growth Medium Kit (C-22110), Medium 2 Kit (C-22111), or Medium MV Kit (C-22120) without adding the ECGS/heparin supplement to the Basal Medium.
Please note: Before and after transfection, the cells should be cultured in complete Growth Medium including heparin to ensure optimal growth.
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Are there PromoCell specialized media / primary human cells alternatives to other supplier’s products?
Please see our attached alternative product guides for media and cells.
Please note: Our media do not contain antibiotics. For optimal cell growth, we recommend to refrain from using antibiotics. However, when a sterile environment cannot be 100% ensured, it may be advisable to add antibiotics to your media so as to protect the cultures from potential microbial infections.
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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-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-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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Does the PromoCell 3D Tumorsphere Medium XF (C-28070) maintain the stem cell character of cancer stem cells?
During our quality control we do not determine the presence or maintenance of respective stem cell markers in the tumorspheres. We use a functional approach instead, by culturing the tumorspheres over serial passages in C-28070. Tumorsphere formation requires the biological features of Anoikis resistance and self-renewal and therefore indicates the presence of cancer stem cells (CSC)/cancer initiating cells (CIC) in the spheres. 3D tumorsphere culture thus allows scientists to study the biology of CSCs without any background knowledge on CSC markers.
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What are the characteristic markers of HDLEC (C-12216, C-12217) and HDBEC (C-12211, C-12225)?
- HDLEC and HDBEC both express the typical endothelial cell marker CD31 (= PECAM-1).
- HDLEC cultures are additionally tested positive for podoplanin, a transmembrane glycoprotein involved in lymphatic vessel formation, whereas HDBECs are podoplanin-negative.
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Can I use the 3D Tumorsphere Medium XF for mouse cell lines?
Yes, we have received a customer feedback that our 3D Tumorsphere Medium XF (C-28070) has been successfully used for tumorsphere formation of mouse cell lines.
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When should I use phenol red-free basal media for cell culture?
It has been shown that phenol red has estrogenic properties. Phenol red-free media are therefore generally used in studies evaluating steroid hormone action in cultured, estrogen-responsive cells (Berthois et al., 1986). Furthermore, phenol red can also interfere with some analytical methods like photometric analyses.
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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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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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What antibiotics concentration should I add to the PromoCell media?
If antibiotics are deemed necessary the recommended final concentrations are:
100 U/ml penicillin + 100 µg/ml streptomycin or
50 µg/ml gentamicin + 50 ng/ml amphotericin BPlease note: Addition of antibiotics can reduce the growth rate of the cells.
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Are the Renal Epithelial Cells isolated from proximal or distal tubuli?
PromoCell provides two types of Renal Epithelial Cells: Human Renal Epithelial Cells (HREpC) and Human Renal Cortical Epithelial Cells (HRCEpC).
HREpC are isolated from the adult kidney and stain positive for cytokeratin. They comprise a heterogeneous population of renal epithelial cells. HRCEpC are isolated from the cortex of the kidney and comprise cells from proximal and distal tubuli. They also stain positive for cytokeratin.Related Links and Documents
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