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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At what passage do PromoCell supply the cell pellets?
The majority of PromoCell cell pellets (C-14**) are prepared 1 passage after thawing the cryopreserved cells.
Examples:
HUVEC and HUAEC correspond to P1 after thawing; therefore the pellets are frozen in P2.
SMC or keratinocytes or epithelial cells correspond to P2 after thawing; therefore the pellets are frozen in P3.
In contrast, our blood cells are cryopreserved directly after cell isolation. These pellets are prepared after thawing the cryovials with no further cultivation step. -
From what tissue do PromoCell isolate their HUtMEC (C-12295)?
Our HUtMEC are isolated from the middle layer of the uterine wall (myometrium). The tissue donors were not pre-treated with hormones.
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What is the composition of the Osteoblast Supplement (C-39615)?
Our Osteoblast Supplement consists of FCS (10 % [v/v] final concentration) which is specifically tested to support optimal growth of Normal Human Osteoblasts.
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Should the skeletal muscle cells be split after adding the Differentiation Medium (C-23061)?
After adding the Skeletal Muscle Cell Differentiation Medium, myoblasts will start to differentiate into myotubes and stop growing. The cells should not be split anymore. It is recommended rather to plate the SkMC into the needed vessels, (e.g. multiwell plates), prior to induction of differentiation and to perform studies directly on differentiated cells. If it is necessary for your tests to detach the myotubes and they are difficult to trypsinze, you can use a “rubber policeman”.
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Does PromoCell offer a special 3-D system to block de-differentiation or does PromoCell have special culture conditions that stabilize the chondrocyte phenotype?
PromoCell Chondrocytes (HCH) can be expanded in normal monolayer culture using our Chondrocyte Growth Medium (C-27101). The Medium consists of an optimized formulation and is supplemented with 10 % FCS. De-differentiation of chondrocytes is a known phenomenon observed during in vitro-culture after a period of approx. 2 weeks, but in vitro-culture is needed to expand the cells.
Once a suitable cell number is obtained, the monolayer system can be changed to a more complex 3-D system either by culturing the cells on substrates like alginate beads, gels, or degradable polymer scaffolds or by using 3-D spheroid culture. Using appropriate conditions, re-differentiation is triggered and the cells start producing cartilage-specific ECM again.
PromoCell does not supply a special culture system but we use 3-D spheroid culture and Alcian Blue staining to characterize our chondrocytes during quality control. The protocol is very similar to the one we use for chondrogenic differentiation of MSC.Related Links and Documents
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Does the PromoCell 3D Tumorsphere Medium XF (C-28070) maintain the stem cell character of cancer stem cells?
During our quality control we do not determine the presence or maintenance of respective stem cell markers in the tumorspheres. We use a functional approach instead, by culturing the tumorspheres over serial passages in C-28070. Tumorsphere formation requires the biological features of Anoikis resistance and self-renewal and therefore indicates the presence of cancer stem cells (CSC)/cancer initiating cells (CIC) in the spheres. 3D tumorsphere culture thus allows scientists to study the biology of CSCs without any background knowledge on CSC markers.
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Does PromoCell have any data regarding the cytokine profile and different markers of the M1/M2 macrophages after activation?
No, we do not provide data about the cytokine profile of our M1/M2 macrophages after activation and we do not provide any further data beyond the scope of our quality control.
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Can the Cytokine Mix E for the Hematopoietic Progenitor Expansion Medium XF (C-39891; 5 ml) be safely aliquoted?
Yes, it is possible to aliquot the 5 ml Cytokine Mix E (C-39891) into 5 x 1 ml.
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Can you control size / size distribution of organoids?
Possibly. The size of the organoids may depend on the access to nutrients in the gel. For reference, https://pubmed.ncbi.nlm.nih.gov/31173716/
For best results we recommend the following:
- Avoid nutrient gradients. We recommend using a small, drop-like BME gel bead and high volumes of medium. Change the medium every day as we’ve observed that less frequent medium changes results in yellow culture medium indicative of a pH shift likely from an increase in cellular metabolism.
- For optimal cell distribution, it is important to control the “gelling” or setting of the ECM gel. Therefore, the temperature is critical! BME is liquid in the cold (4°C) and solidifies at 37°C. The gelling must be done in a very short time, otherwise the cells in the BME would sink to the bottom.
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What is the EXCiPACT™ GMP certification?
Pharmaceutical excipient production should be carried out in accordance with the principles defined in the Good Manufacturing Practices (GMP). With that in mind, a group of industry experts including EFCG, IPEC and PQG worked together to develop the EXCiPACT™ certification scheme for pharmaceutical excipient suppliers.
EXCiPACT™ is a non-profit organization that oversees and operates an independent, high-quality, third-party certification system. This certification system is available to pharmaceutical manufacturers and distributors around the world.
To achieve EXCiPACT™ GMP certification, we demonstrated that our pharmaceutical excipients are manufactured according to the GMP guidelines, as well as ensuring compliance in our raw material supply chain, production environment, production processes and storage conditions.
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I would like to know if the Lymphocyte Separation Medium (C-44010) is endotoxin tested.
Yes, our Lymphocyte Separation Medium 1077 is endotoxin-tested. The specification for the product is < 10EU/ml.
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Is the PromoCell Keratinocyte Growth Medium 2 suitable for growth of primary mouse keratinocytes?
Yes, primary mouse keratinocytes grow well in this media when you reduce the CaCl2 concentration to 0.025 mM – 0.05 mM CaCl2 (optimal Ca concentration for primary human keratinocytes is 0.06 mM).
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When do I need PromoCell’s Monocyte Attachment Medium (C-28051)?
The Monocyte Attachment Medium allows for the efficient adherence selection (within 1 hr) of monocytes from freshly isolated mononuclear cells while maintaining optimal cell health. The Time-consuming and costly immunomagnetic purification of monocytes prior to DC generation is not necessary and can be skipped when using this medium.
.Related Links and Documents
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How long does it take to detach primary cells with accutase (C-41310)?
Most primary cells detach within 5-10 min at 37°C. Inactivation isn’t required but we recommend to centrifuge the cell suspension to remove accutase and EDTA before replating the cells.
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Is it possible to transfect PromoCell Normal Human Cells?
Yes, it is possible to transfect our normal human cells. In general, primary and normal cells are much harder to transfect than immortalized cell lines. Apart from the cell type, successful transfection also depends on the culture’s age and density at transfection, the vector used, the purity of the nucleic acids, the composition of the transfection medium, and the experimental conditions.
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What’s the stability of HPC Expansion Medium XF (C-28021) after addition of Cytokine Mix E (C-39890)?
The medium supplemented with Cytokine Mix E is stable for 2 weeks if stored protected from light at 2-8°C.
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Is it necessary to heat inactivate FCS for use with Normal Human Cells?
The practice of heat inactivation was originally developed when only serum from adult animals was available. Adult serum contains high serum complement which may destroy cells under certain conditions. Heating serum (30 min, 56°C) is intended to inactivate the complement. Today, serum is often heat-inactivated without any evidence of beneficial effect. When using FCS (fetal calf serum), heat inactivation is not necessary for most cell lines or cell types. PromoCell does not use heat-inactivated FCS for the preparation of their growth media.
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To what extent will the CD34+ Progenitor Cells proliferate in Hematopoietic Progenitor Cell Expansion Medium XF (C-28021)?
For optimal cell growth, the human CD34+ Progenitors (C-12921) are cultured in PromoCell Hematopoietic Progenitor Cell Expansion Medium XF (C-28021) supplemented with Cytokine Mix E (C-39890) or with any other cytokine cocktail suitable to achieve optimal cell expansion. Cytokine Mix E contains recombinant human TPO, SCF, flt3-ligand and IL-3. The strong expansion of CD34+ cells will persist for approx. 2 weeks. The expansion factor is usually between 75 and 200-fold.
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What are the criteria for selection or exclusion of peripheral blood donors? Is the peripheral blood that PromoCell use to isolate blood cells (MNC-PB, monocytes) derived from completely healthy donors?
The following criteria are applied for blood donations:
a) Donors with light infection like a cold or cough are excluded for one week
b) Donors with infections with a temperature above 37.9°C and/ or antibiotics therapy are excluded for 4 weeks
c) Blood pressure: exclusion only if systolic blood pressure is < 100 mm Hg or > 180 mm Hg or if diastolic blood pressure is > 100 mm Hg. No exclusion if blood pressure is drug treated and in acceptable range.
d) High cholesterol: no exclusion even though medication is used
e) Diabetes: exclusion if diabetes type I or use of insulin
f) Steroid use: exclusion for 4 weeks after application
g) Cancer: exclusion
h) Other chronic diseases: exclusion depends on the type of disease (for example chronic heart disease, autoimmune disease)Related Links and Documents
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What medium should I use to grow or differentiate PromoCell Human Stem and Blood Cells?
The recommended media for a particular cell type are specified on the respective product page (“Recommended Products”) and can be found in the Manual belonging to the cells.
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From what tissue do PromoCell isolate their HPMEC (C-12281)?
Our HPMEC are isolated from peripheral lung tissue of adult donors.
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How should I thaw the PromoCell normal human cells to obtain a high viability?
General protocol for recovery of anchorage-dependent primary cells:
Remove vial from liquid nitrogen, transport it on dry ice to the cell culture lab. Thaw in a 37°C waterbath for approx. 2 min, until it is just defrosted. Keep the vial immersed in water until just below the screw cap during thawing and only remove it shortly after approx. 90 sec to check the progress.
Carefully disinfect the vial with plenty of 70% EtOH under the laminar flow hood and aseptically transfer the thawed suspension into an appropriate TC dish with growth medium (pre-warmed in the incubator for > 30 min).
The cells usually attach within a few hours. Perform media change after 24 hrs at the latest to remove residual DMSO from the freezing media. Additional information can be found in the instruction manuals of our cells.Related Links and Documents
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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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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.
Related Links and Documents
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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.
Related Links and Documents
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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)
Related Links and Documents
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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.
Related Links and Documents
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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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