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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