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.
Related Links and Documents
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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.
Related Links and Documents
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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.
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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
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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.
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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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How should I seed the preadipocytes for optimal differentiation?
Recommended procedure:
– Thaw the vial according to our protocol
– Seed one part of the cells (5,000 cells/cm²) directly into multiwell plates for subsequent differentiation induction; grow in Preadipocyte Growth Medium until they reach 100% confluency, then induce differentiation according to the recommended protocol
– Seed the other part (5,000 cells/cm²) into a TC dish and expand them in Preadipocyte Growth Medium; trypsinize at subconfluence; if necessary divide the cell suspension again into two parts and use one part for differentiation tests (see above), the other part to continue the culture of undifferentiated HWP.
– Differentiation capacity may decline after 1-2 passages in vitro, therefore best perform your differentiation tests at early passages. HWP at higher passages can still be used for studies that require undifferentiated HWP (e.g. proliferation assays).Related Links and Documents
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What happens if I grow CD34+ or CD133+ progenitors in Hematopoietic Progenitor Cell Expansion Medium XF (C-28021) without Cytokine Mix E (C-39890)?
The cells will die. Hematopoietic Progenitor Cell Expansion Medium XF always must be supplemented with Cytokine Mix E or an appropriate cocktail of cytokines.
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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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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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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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Is it possible to thaw and re-freeze the Cytokine Mix M1 (C-39894) and M2 (C-39895)?
The Cytokine Mix M1 and M2 should not be subjected to further freeze/thaw cycles.
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Can I cultivate the macrophages in complete medium but w/o cytokines?
Without cytokines, the macrophages will die very quickly. You must supplement the Macrophage Base Medium XF (Bsal Medium + SupplementMix) with the appropriate cytokines or at least with human AB ser
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We use Macrophage Generation Media from PromoCell to get differentiated macrophages from peripheral blood monocytes. The monocytes are isolated by culturing PBMCs in Monocyte Attachment Media (also from PromoCell) for 1.5 h. Would it be ok if the attachment step is done overnight, since we receive the blood later in the day which makes it difficult to process on the same day?
It is not recommended to leave the blood cells in the Monocyte Attachment Medium for longer than 1.5-2 hrs. The medium was developed for (short-term) attachment of the monocytes and does not provide nutrients for a longer time period. Leaving the cells in Monocyte Attachment Medium for a longer time or even overnight will induce apoptosis and lead to the loss of the cells.
If necessary, you can reduce the incubation time to 1 hr. In this case, it is advisable to equilibrate the media in the incubator before so that you can immediately and directly add the appropriate amount of PBMC suspension.Related Links and Documents
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Does PromoCell recommend a particular manufacturer or brand of tissue culture plastic to grow the primary cells or can I use any supplier on the market?
Our customers have successfully used TC flasks and dishes from all the leading cell culture plastic suppliers to grow PromoCell’s primary human cells. We do not have any knowledge whether the dishes from local TC plastic suppliers work in the same way. We recommend to first test whether these brands provide the same good performance as the plastic of the leading manufacturers.
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After differentiation of human MSC into mature adipocytes – what medium do I have to use to culture those cells?
Differentiation of hMSC into mature adipocytes takes approx. 2 weeks. You can keep the adipocytes for up to 3 weeks in the MSC Adipogenic Differentiation Medium. After 3 weeks we recommend to switch to PromoCell’s Adipocyte Nutrition Medium (C-27438).
Related Links and Documents
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What is the composition of Melanocyte Growth Medium M2 SupplementMix (C-39420)?
Supplement Mix for Melanocyte Growth Medium M2 (C-39420) is free of serum and doesn’t contain any PMA. The qualitiative and quantitative composition is proprietary.
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What is the source of PromoCell’s Normal Human Follicle Dermal Papilla Cells? For how long can they be maintained in culture?
HFDPC are isolated from the hair papilla of normal human scalp hair follicles. Hair papilla in the adult hair follicle play a crucial role in the dermal-epidermal interactions that control hair production and in hair growth cycle events. The follicle dermal cells are cryopreserved at second passage and can be cultured for at least 10 population doublings when using PromoCell Follicle Dermal Papilla Cell Growth Medium (Cat. C-26501). Typical population doubling times are between 20-36 hrs.
The recommended seeding density after thawing/trypsinization is 5,000-10,000 cells/cm2. Using 1:4 splits, you can perform 4-5 passages with the cells.Related Links and Documents
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Do PromoCell HDMEC exhibit migration and tube formation as a response to angiogenic stimuli?
In principle, all microvascular endothelial cells should be able to migrate, proliferate and form tubes or sprouts in an appropriate assay after angiogenic stimulation. As this is not part of our routine quality control procedure, we cannot tell for sure whether all cell lots will respond to angiogenic stimuli. However PromoCell also supplies HDMEC pre-screened (C-12215) that are especially tested for a positive VEGF response.
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How should I store the PromoCell media and supplements?
Upon arrival, the basal media should be stored between 4°C and 8°C, the supplements at -20°C.
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How does PromoCell characterize their kidney cells (HREpC; HRCEpC) ?
Our kidney cells are characterized by their epithelial cell morphology and by cytokeratin expression using a pan-cytokeratin antibody.
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Which medium do PromoCell recommend to use on HUVEC – Endothelial Cell Growth Medium or Endothelial Cell Growth Medium 2?
The standard medium for isolation and propagation of our HUVEC, HUAEC, HPAEC, and HSaVEC is Endothelial Cell Growth Medium (C-22010). It contains ECGS, an extract from bovine hypothalamus which has mitogenic effects on endothelial cell proliferation. Scientists who prefer a more defined Growth Medium can use Endothelial Cell Growth Medium 2 (C-22011). In this medium, ECGS is replaced by VEGF, IGF, and additional bFGF and EGF to stimulate endothelial cell growth.
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Can I use my own trypsin or other commercially available trypsin solutions for subculturing PromoCell Normal Human Cells?
Most commercially available trypsin solutions have trypsin concentrations of 0.05% or higher which can harm the primary cells. PromoCell recommend using a 0.04% trypsin / 0.03 % EDTA solution for the subculture of Normal Human Cells.
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What plating density should I use for PromoCell Human Adult Stem and/or Blood Cells?
The recommended plating density after thawing/subculture may vary depending on the cell type. It is specified on the Website (tab: additional Information) and in the Product Manual.
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How can I test whether my cells are infected with mycoplasma?
Several different methods for the detection of mycoplasmas have been described, like e.g., cultures on agar, in liquid or semi-solid media, staining with DAPI, mycoplasma-specific antibodies, biochemical methods, and PCR-based assays. PCR-based detection is very sensitive, detects all mycoplasma species that occur in cell cultures and is completed within 3-5 hours.
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Can PromoCell supply preadipocytes (subcutaneous, visceral) from donors with obese BMI / non-smokers / non diabetics? Can PromoCell supply preadipocytes from different age groups?
The basic information we receive from the surgeons about the tissue donors usually includes age, gender, and ethnicity.
- For many of our donors of subcutaneous preadipocytes, we also have information on BMI, hair color, skin pigmentation, and, in some cases, smoking habits or known diseases (e.g. Diabetes). Most of our subcutaneous HWP donors are between ∼20-65 years old.
- The visceral HWP donors are mostly between ∼20-85 years old. For many cell lots we know the BMI, in some cases also the hair color, skin pigmentation, smoking habits, and/or known diseases (e.g. Diabetes or COPD).
If you are looking for particular specifications, please contact our Scientific Support, so that we can offer you appropriate HWP lots.
Related Links and Documents
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Which type of endothelial cell is best suited for studying angiogenesis?
The study of angiogenesis has been significantly advanced by the ability to culture endothelial cells in vitro. Initially, large vessel ECs, such as those isolated from the human umbilical vein (HUVEC) were used for these studies but increasingly it has been recognized that microvascular endothelial cells are a more appropriate model since angiogenesis involves microvessels rather than large vessel ECs.
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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 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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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, including MSC Growth Medium 2.
– 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 or bovine) 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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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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