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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Are the PromoCell Skeletal Muscle Cells (C-12530) in a differentiated or undifferentiated state?
Our SkMC are proliferating myoblasts that have retained the capacity to differentiate. Upon withdrawal of serum and growth factors, differentiation is induced and the cells form multinucleated syncytia.
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After how many passages in monolayer culture do the chondrocytes start to de-differentiate?
Mature chondrocytes from articular cartilage are postmitotic producers of extracellular matrix components like collagen types II, IX, and XI and proteoglycans. Upon release from the tissue of origin and seeding in monolayer culture, cells re-enter the cell cycle and proliferate. After a period of approx. 1-3 weeks, they will gradually start dedifferentiation and will adopt a fibroblastic phenotype. Dedifferentiation can be detected by decreased collagen type II or increased collagen type I expression, as well as by the appearence of the fibroblast marker Thy-1/CD90. Re-differentiation can be induced by changing from conventional monolayer culture to a 3-D culture system.
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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 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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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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Why use ROCKi for organoid formation?
ROCKi is known to enhance the proliferation of epithelial cells or keratinocyte progenitors and can improve cell survival via conditional reprogramming and the effect is reversible. It is also known to enhance the seeding efficiency on plastic.
This ROCKi conditional reprogramming has also been observed in airway EpCs and cultured organoids:
For our human airway organoids, the use of Y-27632 ROCKi is optional as organoids can still form without it. However, the 3D system has some potential pitfalls like choosing the right plastic or the right ECM. The use of ROCKi may help to keep the system more robust.
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Can I access your ISO and EXCiPACT™ Certification?
All our products meet the strictest European and international ethical standards, and our Quality Management System is certified according to ISO 9001:2015 certification and the EXCiPACT™ GMP standard. These certifications ensure that we consistently provide products and services that meet researchers’ and applicable statutory as well as regulatory requirements while also covering the GMP requirements according to NSF/IPEC/ANSI 363.
The following documentations and certification can be directly downloaded on our website
- Quality Policy Statement
- ISO 9001:2015 certificate
- EXCiPACT™ certificate
- ANSI 363-2016 certificate
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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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Looking at the detachment protocol of PromoCell’s Macrophage Detachment Solution, I observed that we need to add HSA to the PBS. Can I use FBS instead? Also, why are we adding 2 mM EDTA to the PBS?
- FBS (or BSA) can be substituted for HSA, however we do not recommend this as the FBS will lead to unpreferable immunologic stimulation of human macrophages.
- The addition of EDTA to the PBS is to further augment the “anti-clumping” activity of the Ca2+/Mg2+ free PBS.
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What is the difference between DC Generation Medium Ready-to-use (C-28050) and DC Base Medium (C-28053)?
- DC Base Medium (C-28053) 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 (C-28050) is ready-to-use and consists of 250 ml Basal Medium + SupplementMix + cytokines.
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Do the PromoCell media contain L-glutamine?
Yes, the specialized PromoCell media already contain the optimal amount of L-glutamine. Please don’t add extra L-glutamine as this can be toxic for the cells.
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What’s the difference between trypsin and accutase?
Both, trypsin and accutase represent mixtures of different proteolytic enzymes. Trypsin is prepared from porcine pancreas, accutase from invertebrates. Accutase can replace trypsin for the detachment and dissociation of anchorage-dependent cells from surfaces and can also be used on suspension cells to reduce clumping in preparation for counting. The advantages of accutase over the traditional trypsin treatment are that it is more gentle and less damaging to cells (leading to increased viability) and does not contain any mammalian or bacterially derived proteins. Accutase is more thermolabile than trypsin and usually doesn’t require an inactivation step.
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Why are most of the soluble receptors not produced in E. coli?
Soluble receptors are post-translationally modified and generally contain disulfide bonds and glycosylation sites. If produced in E. coli the proteins don’t show any biological activity.
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What is the DMSO concentration in Cryo-SFM?
The DMSO concentration in our Cryo-SFM is 10%.
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I am interested in purchasing PromoCell Human Mononuclear Cells (C-12907). Could you please let me know whether you have tested them for platelet contamination? If yes, which markers / procedure have you tried?
During the isolation of our Human Mononuclear Cells (hMNC), we first of all pay attention to thoroughly discard the platelet-containing fraction before we aspirate the MNC containing-interphase of the Ficoll gradient. The harvested MNC fraction is then subjected to several washing steps to remove potential remaining platelets. Finally, the cell preparations are verified by microscopy to be largely free of platelet contamination.
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Can I starve PromoCell HUVECs?
We have many customers who perform starvation with our HUVECs. Most of them use Endothelial Cell Basal Medium supplemented with FCS (0.5-1% for shorter periods; 5-20% for 24-48 hrs). The cells have to be in a good condition and the experiment should be terminated soon after starvation. Prolonged periods will induce apoptosis.
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What media should I use to grow PromoCell Normal Human Cells?
The recommended Growth Media are specified in the Manual belonging to the cells and can be also found on the product page of the cells (“Recommender products”).
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What are the precise localizations of both, juvenile and adult HDMEC?
Juvenile HDMEC (C-12210) are isolated from the dermis of foreskin. Adult HDMEC (C-12212) are isolated from different regions. The localizations include cheek, temple, breast, upper arm, and labia. Please contact our Technical Customer Support if you need HDMEC from a particular part of the body.
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My Normal Human Cells have changed their morphology. What could be the reason?
Your observation can be either ascribed to a change of the culture medium, to a cross-contamination with another cell type, or to differentiation or senescence. The attached trouble shooting guide should help you to identify the reason for your observation.
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Can I mix the SkMC Supplement with the Skeletal Muscle Cell Basal Medium and freeze some down for later use? If so, how long can it remain frozen?
The PromoCell Basal Media must be stored between 4-8°C and should not be frozen, as this can lead to precipitations. The same is true after addition of the supplements: the complete medium has to be kept at 4-8°C.
If you prefer to make up smaller volumes of complete medium, you can aliquot the Supplement Mix and refreeze those aliquots at -20°C until use. This way you can extend the period in which you can use the supplemented media.Related Links and Documents
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When we buy chondrocytes from PromoCell, is it possible to make sure they are articular chondrocytes, i.e. from knee joint?
All our chondrocytes (C-12710) are isolated either from knee joint or femoral head. If you need chondrocytes specifically from either localization, please contact our Technical Customer Service before placing your order. They will send you the available lot numbers from the localization that you are looking for.
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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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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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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 do you set up high throughput systems with organoids? Only in 96 well?
It is possible to culture the airway organoids in 96-well U-Bottom plates for suspension cells (e.g., Greiner Bio-One # 650185).
A detailed protocol for the use of 96-well plates can be found in our AppNote. We have not tested 384-well plates or other commercially available plates.Related Links and Documents
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Where can I find the supporting documents and the material safety data sheet for your products?
Using the product lot number the Supporting Documents can be downloaded at: www.promocell.com/eq-certificates
The Material Safety Data Sheet (MSDS) can be directly downloaded from our website on the product page”
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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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