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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In the past, we used MSC Growth Medium (C-28010) in our lab. Can I use MSC Growth Medium 2 (C-28009) instead? What is the difference?
MSC Growth Medium 2 (C-28009) is an optimized medium formulation with reduced serum content to allow for more standardized culture conditions (considerably lower lot to lot variation).
You can replace MSC Growth Medium by MSC Growth Medium 2. Coating of culture vessels is not necessary. We recommend to plate the cells (hMSCs from bone marrow, adipose tissue, or umbilical cord) at 4,000 cells/cm².Related Links and Documents
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Does PromoCell recommend a specific plasticware to use for the generation of dendritic cells from peripheral blood monocytes?
Usually, we do not recommend specific plasticware but we have found that with PromoCell’s Dendritic Cell and Macrophage Generation Media, choice of plasticware can have a lot of an influence. For our Dendritic Cell Generation Media we recommend to use tissue culture vessels from BD FalconTM.
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What is the difference between DC Generation Medium XF (C-28052) and DC Base Medium XF (C-28054)?
- DC Base Medium XF (C-28054) consists of 250 ml Basal Medium + SupplementMix. It does not include cytokines and must therefore be supplemented according to the user’s individual needs.
- DC Generation Medium XF (C-28052) is ready-to-use and consists of 250 ml Basal Medium + SupplementMix + appropriate cytokines.
Both, Base Medium XF and Generation Medium XF have a xeno-free formulation.
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What’s the difference between PromoCell HUVECs and HUV-EC-C from ATCC?
PromoCell HUVECs are freshly isolated from umbilical veins. They are cryopreserved at the end of primary culture. After revival, they can be propagated for at least 15 doublings and will senesce eventually.
HUV-EC-C from ATCC is a hypodiploid human cell line of endothelial origin (umbilical vein). The modal chromosome number is 45 occurring in 72% of cells counted. The rate of polyploid cells is 15.8%. The cells have a life expectancy of 50 to 60 population doublings. This indicates that the cells are no longer “normal cells” but have undergone some degree of transformation.Related Links and Documents
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Will the hematopoietic progenitor cells proliferate in Hematopoietic Progenitor Cell Expansion Medium XF (C-28021) without Cytokine Mix E (C-39890)?
For the expansion of hematopoietic progenitors (CD133+ cells, CD34+ cells), PromoCell has developed Hematopoietic Progenitor Cell Expansion Medium XF (C-28021), a serum-free and xeno-free formulation. The medium must be supplemented with either Cytokine Mix E (C-39890) or with the user’s own mixture of cytokines. Cytokine Mix E is a ready-to-use mix containing recombinant human TPO, SCF, flt3-ligand, and IL-3.
The strong expansion of the progenitor cells in Expansion Medium XF + Cytokine Mix E persists for at least two weeks, resulting in a 200-300x increase of the cell number with only 20-30% differentiated cells.
Note: If starting with CD133+ cells, the CD133 marker is getting lost during this expansion step. The resulting cells are D34+/CD38–/CD133–.Related Links and Documents
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Can mycoplasma contamination be observed with the naked eye?
No, mycoplasma can only be observed through electron microscopy. For highly sensitive detection of mycoplasma contamination, we recommend the use of PCR-based mycoplasma tests.
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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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How many vials of MNC-PB can PromoCell typically provide from one lot/one donor?
There is a strong variation from donor to donor and sometimes from donation to donation concerning the number of vials that can be produced. This is due to individual variances between the donors as some of them have more MNCs per ml of blood and others have less.
Generally, the number of vials (each with 25 x 106 cryopreserved cells) per lot ranges between 10 and 40.Related Links and Documents
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Is it possible to mineralize PromoCell osteoblasts?
Yes, it is possible.
Short protocol: Plate human osteoblasts in Osteoblast Mineralization Medium on collagen I coated TC vessels. Incubate the cells for 17-21 days and change the medium every third day. Be careful not to disturb the cell monolayer. Fix the cells. The calcium deposition can be visualized by von Kossa or Alizarin Red staining.
More detailed information on osteoblast mineralization and Alizarin Red S staining can be found in the attached Application Note.Related Links and Documents
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Can I use accutase solution instead of trypsin to detach the cells?
Yes, you can use accutase to detach Normal Human Cells. Accutase acts very gently on the cells. Cell membranes and surface epitopes will not be harmed. It is therefore mostly used for applications that require unchanged surface markers, e.g. for flow cytometry, or for detachment of very sensitive cells.
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What is the composition of PromoCell Chondrocyte Growth Medium?
Our Chondrocyte Growth Medium consists of an optimized basal media formulation and is supplemented with 10 % (v/v) fetal calf serum that has undergone stringent biological controls.
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My cells are contaminated. Where does the contamination come from?
Microbiological contaminations can be introduced into cell cultures by unsterile working techniques, contaminated water baths, media, plasticware, etc. Please check the attached trouble shooting guide for more detailed information.
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At what level of confluency should the SkMC differentiation typically be initiated?
We have obtained best results when the cells have reached 60-80 % confluency. At this point, the Growth Medium is aspirated and replaced by Skeletal Muscle Differentiation Medium. After 2 – 8 days, extensive formation of multinucleated syncytia can be observed. For a stable differentiation of SkMC switch back to Skeletal Muscle Cell Growth Medium after 5 days incubation in Skeletal Muscle Differentiation Medium.
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Do you get new blood samples in every few days for MNC-PB as well as MNC-CB isolation so that a good variety and number of cells are available to suit our purchase needs?
We get 5-8 new lots of adult MNC (C-12907) every month (> 15 vials each). Cord blood MNC (C-12901) are smaller lots (5-10 vials), we get 2-4 new lots every month.
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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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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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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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Do you find that the domes spread a lot on the Nunclon Sphera plates? Do they remain attached for the entire 3-4 week culture period?
To avoid the spreading of the matrix and to have nice drop-like domes, it is crucial to preheat the plate for 2h at 37°C and to work fast when transferring the gel to the wells. Placing a warming plate under your laminar air flow hood when transferring the gel-cell-mix to the preheated plate may help.
From our testing, we found that Nunclon Sphera wells were not ideal for our organoid system as the dome did not stay adhered, but it was able to move on the bottom and the dome became misshaped overtime. However, the altered shape of the dome did not affect the organoids and were successfully cultured for 4 weeks. If movement is not an issue for the user, we recommend that extra caution be taken during media changes by holding the plate at an angle and carefully pipette to remove old medium. Aspirating with vacuum suction can damage or destroy the dome.
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What is the limitation for the intention of use for your products?
- Our standard research products are intended for in vitro research use only.
- Our GMP-compliant and regulated media are intended for research use or further manufacturing. They are not intended for direct administration to humans or animals.
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Is it possible to carry out the macrophage differentiation in either a 96- or 384-well plate, so long as the volumes are adjusted accordingly? If not, is it possible for cells to be detached and re-plated in the desired culture plate?
a) We usually perform macrophage differentiation in T75 flasks and 6-well plates. We haven’t tested differentiation in smaller formats. But we assume it will be problematic to thoroughly wash the surface of the wells to remove non-adherent cells after the attachment phase. b) Detachment of the mature macrophages is possible but re-attachment can lead to significant cell loss (30-50%).
Please also keep in mind that working in 96/384 well-format has some inherent drawbacks (e.g., evaporation of media, dry wells, etc.).Related Links and Documents
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I was wondering if you had any suggestions regarding the plasticware to use for monocyte enrichment/macrophage differentiation.
Usually, we do not recommend specific plasticware but we have found that with PromoCell’s Dendritic Cell and Macrophage Generation Media, choice of plasticware can have a lot of an influence.
For our M1-/M2 Macrophage Generation Media XF we recommend the Nunc plasticware with Nunclon surface – as not only will the detachment efficiency vary (up to 20%), but also the efficiency of the differentiation process itself may be altered.
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What is the pH of your Endothelial Cell Culture Media MV2?
The pH of our Endothelial Cell Basal Media MV/MV2 is 7.4 ± 0.1.
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What’s the difference between PromoCell HUVEC single donor (C-12200) and HUVEC-pooled (C-12203)?
Our HUVEC single donor (C-12200) are isolated from a single umbilical cord, propagated in primary culture, and frozen down at subconfluency.
For the preparation of HUVEC-pooled (C12203), we simultaneously isolate the cells from 2-4 umbilical cords and grow them in separate tissue culture dishes. The cells are pooled after trypsinization given that their growth rates are comparable.
After thawing, our HUVECs (single donor and pooled) are both in P1. The recommended media are Endothelial Cell Growth Medium (C-22010) or Endothelial Cell Growth Medium 2 (C-22011). With respect to cell growth, HUVEC-pooled tend to have a more heterogeneous morphology with slightly more elongated cells but the doubling times are comparabel for both types (typically 18-36 hrs per doubling).Related Links and Documents
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What is the exact localization of PromoCell’s HAoAF (Human Aortic Adventitial Fibroblasts)?
Our HAoAF (C-12380) are isolated from the Adventitia, the outer layer of the aorta. The cells have been characterized as fibroblasts by the expression of fibroblast-specific CD90.
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At what passage are PromoCell Mesenchymal Stem Cells / Pericytes upon arrival?
Our Human Mesenchymal Stem Cells and Human Pericytes are cryopreserved at the end of secondary culture (P1). After thawing, they are in P2.
Related Links and Documents
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Do I need additional supplements when working with PromoCell growth media? Do I have to add extra FCS?
After addition of the SupplementMix or SupplementPack to our basal medium, you obtain the complete growth medium. No further supplementation with serum or growth factors is required. Please note: Our media do not contain antibiotics. If you wish to use antibiotics you can add penicillin/streptomycin or gentamicin/amphotericin B at standard concentrations. Addition of antibiotics can however reduce the doubling time of the cells up to 30-40%.
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What can pericytes differentiate into?
Pericytes have been shown to differentiate e.g. into adipocytes, osteoblasts, chondrocytes, fibroblasts/myofibroblasts, vascular smooth muscle cells, and phagocytes.
Related Links and Documents
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How many passages can I perform with PromoCell HUVECs?
PromoCell guarantee > 15 PD for their HUVEC. The number of passages that can be performed, depends on the dilution factor used during subculture. If you split the cells 1:4, they can perform about 2 population doublings per passage which means that they can be cultured for at least 6-8 passages.
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Can I trypsinize the PromoCell Normal Human Cells at 37°C?
PromoCell recommend to trypsinize all Normal Human Cells at room temperature and to monitor the detachment under the microscope. Prolonged trypsinization at 37°C can lead to irreversible damage of the cells.
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How do I best store PromoCell cell pellets? What is their shelf life?
PromoCell cell pellets (C-14**) can be stored indefinitely at -20°C and are stable for up to 1 month at 4°C and up to one week at room temperature.
Please note: The samples do not freeze at -20°C.
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My Normal Human Cells stopped growing and died after a few weeks in culture. What could be the reason?
Normal cells have a finite life span and therefore eventually stop growing and become senescent. In addition, a cease in proliferation or cell death can be induced by other factors. For more details, check the trouble shooting guide below.
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How can the SkMC be differentiated? Can you send me a protocol?
To differentiate the SkMC into myotubes, we recommend to use cells that have undergone a maximum of 4-5 population doublings.
For this, the cells (ideally in P2 or P3) are cultured to 60-80% confluency in Skeletal Muscle Cell Growth Medium (C-23060). Then, a change to (serum-free) Differentiation Medium (C-23061) is performed to induce the differentiation process (formation of multinucleated syncytia). After 5 days, switch back to the Skeletal Muscle Cell Growth Medium for another 8 days to complete the differentiation.
This protocol leads to stable myotubes and some of the myotubes show spontaneous contractions.Related Links and Documents
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The Certificate of Analysis of hMNC contains the percentages of the main cell types (lymphocytes, monocytes, …). How does PromoCell determine that these are monocytes?
Our MNC lots are checked for the rate of lymphocytes, monocytes and granulocytes w/o staining by FSC/SSC using a flow cytometer. A sample plot is attached. For our purified CD14+ monocytes we additionally perform a CD14+ staining.
Related Links and Documents
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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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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.
Related Links and Documents
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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.
Related Links and Documents
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Can I use a bead bath to thaw PromoCell normal human cells?
Based on negative feedback we have received from customers using bead baths, we strongly discourage the use of bead baths to thaw our cells. It can lead to reduced viability or significantly slower growing cells.
If you don’t have a “normal” water bath but only a bead bath in your lab, thaw the vial in a beaker of water in the bead bath. Ensure the water is heated to exactly 37°C using a thermometer placed in the warmed water. Be sure to hold the vial in your hand, and not in a floater, as described in the thawing protocol.
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How do you define your media & reagents specifications?
Choosing a suitable culture medium is crucial factor for in vitro cell cultivation and significantly affects the success of cell culture experiments, from the first step of development and when transitioning to clinical applications. Due to specific requirements of primary cells and each researcher’s application, we provide a wide range of advanced media formulations.
Therefore, it is essential for researchers to have a clear understanding of how to define the specifications of our cell culture media and reagents, from serum-free or xeno-free to chemically defined, to estimate and understand the associated implications for their intended applications. We can support you by providing a comprehensive guideline for the characterization and qualification of our cell culture media and reagents.
Discover and download our Media & Reagents Specification Guide
Related Links and Documents
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What are the advantages of monocyte enrichment by adherence selection using your Monocyte Attachment Media (C-28051)?
This method produces large numbers of adherent monocytes in only 1.5 h. If the washing steps are properly performed, 80-90% purity can be expected. The attached cells are “untouched”, since no binding of magnetic microbeads has occured. This also excludes phagocytosis of the microbeads by the monocytes, an event which is unfavorable with regard to cellular health. In addition, the adherence method is time-saving and cost-effective.
Related Links and Documents
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Is it normal that the cell pellets from PromoCell (C-14**) do not freeze at -20°C?
Samples in RNAlater will NOT freeze at -20°C, they are liquid at 4°C and -20°C. The samples can be stored at 4°C for up to 1 month and are stable at room temperature for up to one week. At -20°C, they can be stored indefinitely.
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What’s the difference between NHEK.f and NHEK.f pooled?
NHEK.f are isolated from tissue samples (foreskin) of single donors, aged between 1-10 years. NHEK.f pooled are prepared from the foreskins of 3 individual donors. The cells of each donor are expanded in separate TC vessels and the cells are pooled after secondary culture, before cryopreservation.
Both NHEK from single donor and NHEK pooled are in P2 after thawing.Related Links and Documents
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Do the PromoCell keratinocytes need feeder cells?
If you are growing our NHEK in PromoCell Keratinocyte Growth Medium 2 (C-20011) or Growth Medium 3 (C-20021), you don’t need any feeder cells. The cells will grow as a monolayer in conventional tissue culture flasks.
Related Links and Documents
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Why does PromoCell only guarantee 15 PDs when with my own self isolated dermal fibroblasts I regularly achieve 30 passages?
The PromoCell quality control procedure includes the cultivation of fibroblasts for 15 population doublings. This is to ensure that the cells can be grown for a minimum of 15 PDs (6-8 passages depending on the split ratio used) but it does not mean that the cells immediately senesce after that point. We don’t determine the maximum number of doublings or passages but most NHDF lots will certainly achieve > 20 passages.
Related Links and Documents
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What is the reason for turbid serum?
Serum turbidity is usually caused by cryoprecipitation of lipid components during freezing and thawing. The more times the serum is subjected to freeze/thaw cycles, the more turbidity is noticed. It can be minimized by freezing the serum in aliquots at -20°C and thawing these aliquots individually at the time of use.
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Can I aliquot the SupplementMix?
Yes, you can aliquot the SupplementMix upon delivery and freeze down 2 or 4 individual aliquots at -20°C. This way, you can prepare smaller volumes (2 x 250 ml or 4 x 125 ml) of complete culture medium and thus extend the time you can use the medium.
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Why does PromoCell use CD146 as a marker for pericytes?
CD146 (MUC18) is a surface marker, expressed on pericytes, MSCs, and endothelial cells from large vessels (but not microvascular endothelial cells). In combination with CD34 (a marker for endothelial and hematopoietic cells), pericytes can be characterized by FACS analysis as CD146+/CD34–. The absence of CD34 expression makes sure that the cells are not of hematopoietic/endothelial origin. Further markers that have been described for pericytes are NG2, CD90, alpha-SMA, and PDGFR-beta.
Related Links and Documents
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Are the HDMEC pre-screened (C-12215) derived from juvenile foreskin or from adult skin?
All HDMEC pre-screened lots that we currently have in stock are isolated from foreskin.
If you need pre-screened HDMEC from adult donors, please contact the PromoCell Technical Customer Support.Related Links and Documents
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Is it possible to perform experimental starvation with PromoCell Normal Human Cells and the PromoCell media?
Yes, but the experimental starvation conditions have to be determined individually for each cell type. Usually, the cells are maintained in basal medium with reduced growth factor concentrations or lower FBS content. The cells need to be in a good condition and the starvation should be kept as short as possible as prolonged serum and/or growth factor deprivation induces apoptosis.
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At what passage are PromoCell Normal Human Cells upon arrival?
The passage number varies depending on the cell type. Please refer to the Manual for your cells under “Specifications”.
You will also find the information in the Certificate of Analysis (CoA).Related Links and Documents
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My Normal Human Cells are growing slowly after subculture. What could be the reason?
Slow growth after subculture can be caused by over-trypsinization or other suboptimal culture conditions. Please see attached trouble shooting guide for possible reasons.
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