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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Could you please let me know whether you have used the baculovirus expression system to produce components of your media?
Baculovirus is generally used in conjunction with insect cells (Sf-9, Sf-21) to produce recombinant proteins (cytokines, growth factors).
None of our Specialized Media (Media for Primary Human Cells; Blood and Stem Cell Media, Cancer Cell Media) contain recombinant proteins produced in insect cells. This also applies to our Cryo-SFM Freezing Medium. -
Do I need precoated flasks when growing my MSC in PromoCell Mesenchymal Stem Cell Growth Medium XF (C-28019)?
Our MSC Growth Medium XF provides a xeno-free culture system for human MSCs. It contains all growth factors and supplements except attachment- and spreading factors.
Therefore, culture vessels to be used with Mesenchymal Stem Cell Growth Medium XF must be precoated either with 1 μg/cm2 human fibronectin or 0.5 μg/cm2 human vitronectin according to the instruction manual of the manufacturer.
Alternatively, bovine fibronectin may be used.Related Links and Documents
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Is it normal that CD34+ progenitor cells attach to tissue culture plastic after a week-long culture in HPC Expansion Medium XF (C-28021)?
The cells sink down to the bottom of the culture vessel but don’t really attach. They retain a roundish morphology and can be rinsed off with culture medium easily.
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How should I handle PromoCell’s proliferating cells after arrival?
Short description:
Unpack the box and place the T25 flask(s) in the incubator for 3 hrs (closed cap). Then check confluency under the microscope.
When the density is < 70%, aspirate the medium using sterile conditions and add 5-10 ml of the appropriate Growth Medium. The cells should be subcultured according to the subcultivation protocol given in the cells’ Instruction Manual once they have reached > 70 % confluency.Related Links and Documents
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I have a question related to PromoCell’s CD34+ Progenitor Cells (C-12921). Are they suspension cells or do they partially attach?
It actually depends on the culture conditions whether the cells remain in suspension or attach to the surface.
When grown in our serum-free, xeno-free HPC Expansion Medium XF (C-28021), the cells remain in suspension.Related Links and Documents
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Can rat or mouse SkMCs be cultured using the Skeletal Muscle Cell Growth Medium (C-23060)?
Yes, the Skeletal Muscle Cell Growth Medium can also be used for rat, mouse and rabbit SkMC.
We recommend to use the medium right after isolation. Cells that were isolated and cultured in a different medium beforehand may have adapted to the other medium. An abrupt medium change causes stress to the cells resulting in reduced growth rates and lower differentiation capacities.Related Links and Documents
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When should I order the Growth Medium Kit instead of the Medium “ready-to-use”?
The Growth Medium Kit allows customization of the end concentrations of growth supplements. It is therefore more flexible than the Medium “ready-to-use” and can eg. be used to prepare a starvation medium.
Please note: Modification of supplement concentrations may have an impact on cell growth. You should test in advance whether and for how long the cells can survive the altered culture conditions.Related Links and Documents
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I would like to know how homogenous the MSC population is and if all cells become osteoblasts when grown in MSC Osteogenic Differentiation Medium (C-28013).
Human Mesenchymal Stem Cells have retained the ability to differentiate into a variety of cell types including fat cells, chondrocytes, and osteoblasts. PromoCell supply hMSC from 3 different tissues: bone marrow (hMSC-BM; C-12974), umbilical cord matrix (hMSC-UC; C-12971), and adipose tissue (hMSC-AT; C-12977).
- MSC-BM show very good differentiation into bone cells but also into chondrocytes and fat cells when the respective Differentiation Media is used.
- MSC-UC have a high potential to differentiate into chondrocytes but only weak potential for fat or bone cell differentiation.
- In contrast, MSC-AT differentiate very well into fat and bone cells but only moderately into chondrocytes.
In other words, to obtain a high percentage of bone cells, MSC-BM or MSC-AT are the cells of choice. There are of course lot-to-lot variations and the differentiation will decrease from passage to passage. If you need MSCs with a particularly high osteoblast differentiation capacity, you can call our Technical Customer Service before placing your order so that we can select an appropriate cell lot for you.
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Can PromoCell normal human cells be grown in standard media like DMEM or RPMI1640 supplemented with 10% FBS?
PromoCell Growth Media have special formulations and are much more complex than DMEM or RPMI + FBS. In comparison to immortalized cell lines, primary cells have much higher nutrient and growth factor requirements. Classical media do not usually achieve good performances with our cells.
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Can I isolate RNA and/or proteins from the cell pellets?
PromoCell cell pellets (C-14**) are an easily accessible source of DNA, RNA, and proteins.
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How many population doublings do PromoCell guarantee for the Normal Human Cells?
PromoCell guarantee 15 population doublings (PD) for most Normal Human Cells (unless otherwise indicated on the Certificate of Analysis) when the recommended PromoCell media and the PromoCell DetachKit are used. For more information, please check the respective Manual, section “Specifications”.
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Can I first expand the osteoblasts and then perform my experiments?
Normal osteoblasts, similar to other non-transformed cell types can be expanded in vitro to a certain extent before they are used for experiments. Nonetheless, HOB are generally used at low passages (up to P4) in most labs.
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Does the Chondrocyte SupplementMix (C-39635) contain any growth factors?
The Chondrocyte Growth Medium SupplementMix consists of Fetal Calf Serum (final concentration 10 % v/v) which has specifically been tested for the culture of primary chondrocytes. FCS contains a variety of different growth factors, which are however, not analyzed in more detail. There are no further growth factors added to the SupplementMix.
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Where does PromoCell source the bronchial tissue for HBEpC preparation? Are the smoking habits of the donors known?
We source the bronchial tissue from forensic medicine and from thoracic surgery. For some lots, the smoking habits of the donors are known.
Please contact our Technical Customer Service before ordering the cells if you need this information.Related Links and Documents
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Is vWF a great marker for Endothelial Cells?
Secretory granules called Weibel-Palade bodies (WPBs) containing Von Willebrand factor (vWF) are more linked to the formation of a confluent endothelial monolayer. Research shows that vWF expression is dynamic and highly dependent on the cell culture conditions such as confluence and passage number.
Therefore, having vWF as a quality control marker for each lot of ECs is not really necessary.Related Links and Documents
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I would like to differentiate hMSC into bone cells and further maintain the cells in culture after differentiation. Which medium should I use?
Osteogenic differentiation of hMSCs with PromoCell MSC Osteogenic Differentiation Medium takes ∼12-14 days (please view the respective Application Note for a detailed protocol). Use fibronectin-coated plates and change the medium every third day.
The bone cells tend to detach from the plastic after approximately 2 weeks, when differentiation is complete. Therefore, the tests should be performed promptly and the cells should be maintained in MSC Osteogenic Differentiation Medium until then.
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Are your NHEK from the epidermis of the juvenile foreskin mucosal or cutaneous keratinocytes or a mixed population?
PromoCell’s Normal Human Epidermal Keratinocytes (NHEK) from juvenile donors are isolated from both the epidermis of the outer and mucosal (i.e. inner) layers of the foreskin. Thus, each vial contains a mixture of mucosal and cutaneous keratinocytes.
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I would like to know if, in addition to HBEpC, the other airway epithelial cells are also compatible with PromoCell ALI medium.
- For our HNEpC and HTEpC, please refer to the instructions in the AppNote describing the differentiation of these cell types using our ALI medium.
- We do not provide instructions for HSAEpC because our medium is not suitable for differentiation of this cell type at the air-liquid interface.
- For our HBEpC we have special ALI prescreened lots in stock that were successfully tested for barrier function in our QC. Please contact our technical customer support for more information.
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For how long can the monocyte-derived DCs be maintained in culture after the differentiation is completed (day 7/8 of the protocol)?
According to our experience, they can be maintained for 3 to a maximum of 7 days. However, the morphology will change and they will look more and more "degenerate".
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How big is the portion of MSC that can be differentiated into neuronal lineages?
Usually 90-100% of the hMSC show a neuronal morphology after differentiation with our Mesenchymal Stem Cell Neurogenic Differentiation Medium (C-28015). 60-80% of them are positive for nissl bodies after a nissl stain. However, the differentiation capacity depends on the origin of the cells and the number of population doublings they have undergone.
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What is the difference between PromoCell’s HREpC and HRCEpC?
Our HREpC (C-12665) comprise a heterogenous mixture of renal epithelial cells isolated from cortex and medulla. The HRCEpC (C-12660) are derived from renal cortex only.
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What are the different types of multiwell plates that can be used in fluorescence, luminescence and colorimetric detection? Which type should I use for which application?
1) For fluorescence detection (fluorometer): Black plates with clear bottoms; often clear plates will suffice 2) For luminescence detection (luminometer): White/opaque plates 3) For colorimetric detection (photometer): Clear plates
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Which of the PromoCell DetachKits, C-41200 or C-41202 should I use to trypsinize the HNEpC?
You can use our standard DetachKit (C-41200) to subculture the human nasal epithelial cells. Some customers still prefer to use our DetachKit-2 (C-41202) as it has a lower trypsin/EDTA concentration but tests in our cell culture lab haven’t revealed any adverse effects when using C-41200.
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From what muscles does PromoCell isolate the SkMC?
Our skeletal muscle cells are mainly isolated from M. pectoralis, sometimes also from M. gastrocnemius, M. intercostales or M. gluteus maximus.
The exact localization is specified in the Certificate of Analysis.Related Links and Documents
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When should I use DetachKit, when DetachKit-2?
Our DetachKit (C-41200) is optimized for primary cells and can be used to trypsinize all Normal Human Cells supplied by PromoCell.
Detach Kit-2 was developed for very gentle detachment and is still used by some investigators to subculture Human Nasal Epithelial Cells (HNEpC).Related Links and Documents
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Can I detect the differentiation of Mesenchymal Stem Cells by their morphology, without doing any cell staining?
- Adipogenic differentiation can be identified morphologically and without any staining by the formation of intracellular lipid vesicles.
- In contrast, when MSC differentiate into bone cells, there is no significant change in morphology. It is recommended to perform Alkaline Phosphatase staining to detect osteoblastic differentiation or Alizarin Red S staining to show osteoblast mineralization.
- Chondrogenic differentiation is generally performed as spheroids in 3-D cell culture and not in 2-D monolayer culture. Staining with Alcian Blue to visualize the differentiation process is indispensable.
- Neurogenic differentiation can be detected using neuron specific markers (e.g. beta-3 tubulin, NeuN, MAP2) and by their typical neuronal morphology.
For differentiation protocols, please see attached Application Notes.
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What markers does PromoCell test to characterize their Adult Human Stem and Blood Cells?
All Adult Human Blood and Stem cells are analysed by flow cytometry to express defined markers. MSCs are additionally tested for their capacity to differentiate into the osteogenic, chondrogenic and adipogenic lineages. For more details, please see Certificate of Analysis of the respective cell type (“Phenotypic characterization”).
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Can the cell pellets be used to establish a growing culture?
No, the PromoCell cell pellets (C-14**) are frozen at -20°C and cannot be revived. Their main application is to analyze RNA or protein. Cryopreserved cells that can be revived are available from the same donors. Please contact our Technical Customer Support if you need matched viable cells.
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How many passages can I perform with PromoCell Normal Human Cells?
PromoCell does not determine the number of passages but instead we calculate the population doublings (PD) that can be performed with the cells. The term passage only describes the process of detachment and replating and does not take into account different split ratios. The optimal split ratio is calculated from the actual cell yield after trypsinisation and the recommended plating density. In most of our cell types, the split ratio is usually between 1:3 and 1:6. Using 1:4 splits (i.e. increasing the growth surface by factor 4 each time), 15 doublings are achieved after 6-8 passages.
For recommended plating densities, please view the respective Manual, section “Specifications”. -
Does the Osteoblast Growth Medium support osteoblast proliferation or differentiation? Does the medium contain any recombinant growth factors?
Our Osteoblast Growth Medium (C-27001) consists of the Basal Medium supplemented with 10% (v/v) FCS, but with no recombinant growth factors. The medium primarily supports the proliferative capacity of normal human osteoblasts. It does not contain osteogenic factors (like dexamethasone and beta-glycerophosphate) that promote differentiation as many users test their own chemical compounds (growth factors, hormones), or examine the effects of physical strain or sheer stress on the differentiated functions. The Osteoblast Growth Medium is well-suited as the basis for these applications and can be supplemented with further growth factors if necessary. To specifically induce mineralization, PromoCell supply Osteoblast Mineralization Medium (C-27020).
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Is it feasible to store the melanocytes for a second time in liquid nitrogen after subculture, similar to cell lines?
Upon arrival, you can either defrost the cells immediately and seed them in a TC vessel or store the vial in liquid nitrogen until needed.
It is possible to re-freeze normal human cells but PromoCell does not recommend it, because each freezing cycle leads to a loss in proliferation potential. If you want to freeze them, please use our Cryo-SFM (C-29910). It is also recommended to freeze the cells at an early passage.
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What is the exact localization of PromoCell’s HNEpC (C-12620)?
Our Nasal Epithelial Cells are isolated from nasal septum or adenoids.
Related Links and Documents
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Are endothelial cells α-SMA-negative under all circumstances?
Scientific findings from different groups, as well as our own results indicate that the presence or absence of α-SMA is not a valid indicator for the composition of a given cell population. Research findings and our inhouse data suggest that endothelial cells are not α-SMA-negative under all circumstances. α-SMA negativity is not an intrinsic property of endothelial cells but can vary depending on extrinsic influences.
Lot-specific values for α-SMA in our Certificates of Analysis (CoAs) are therefore not considered meaningful.
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Which human AB serum does PromoCell recommend to generate M0 macrophages?
We recommend to use Human Serum AB „off-the-clot”.
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Can your Macrophage Detachment Solution also be used for bone marrow-derived macrophages from mice?
Yes, there is a reference (Campuzano et al.; J Immunol. 2020 Jun 15;204(12):3296) where bone marrow-derived macrophages from mouse were detached using our Macrophage Detachment Solution (40 minutes at 4°C).
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Can I use frozen PBMCs instead of CD14+ monocytes to generate dendritic cells with PromoCell DC Generation Medium?
PBMCs (= peripheral blood mononuclear cells) consist mainly of lymphocytes and monocytes. Cryopreservation causes the CD14+ monocytes to significantly lose their ability to attach to TC plastic. Therefore, the use of frozen PBMCs as starting material for DC generation is not possible because the purification step with Monocyte Attachment Medium will not work.
You can either start with cryopreserved CD14+ monocytes (C-12909) or use freshly isolated mononuclear cells or fresh CD14+ monocytes.
Please refer to the Product Manual of C-28050 or our Application Note [Generation of monocyte-derived Dendritic Cells] for the appropriate protocol.Related Links and Documents
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From which part of the leg does PromoCell isolate the HSaVEC?
The Vena saphena section that we use for HSaVEC isolation originates from the thigh.
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How long can I keep neuronally differentiated MSC in culture?
Changing the medium every 2-3 days, the neuronally differentiated MSC can be kept in culture for up to 2 weeks.
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What population doubling times can I expect when growing PromoCell’s Mesenchymal Stem Cells? How long does it take from seeding to subculture?
The population doubling times for PromoCell hMSC (hMSC-BM, hMSC-AT, hMSC-UC) are typically ≤ 30 hrs when using Mesenchymal Stem Cell Growth Medium 2 (C-28009).
If you seed the hMSC at 4.000 cells/cm2, it will take between 4-7 days until they reach subconfluency.Related Links and Documents
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What’s the difference between visceral and subcutaneous HWP? When should I use which cell type?
PromoCell Preadipocytes are isolated from subcutaneous or visceral fat.
Subcutaneous fat is found just underneath the skin and it is not related to the obesity-linked diseases. Its accumulation represents the normal physiological buffer for excess energy intake. When the storage capacity of subcutaneous fat is exceeded or the generation of new adipocytes is impaired, visceral fat starts to accumulate. It is located in the abdomen and around internal organs (e.g. kidney, heart, or bladder) and it is linked to hypertension, diabetes, and cardiovascular disease.
Adipocytes from subcutaneous and visceral fat differ in several functions, like their response to insulin and other hormones or their lipolytic activity. It highly depends on the scientific problem being addressed, which of the two cell types are best suited for your experiments.Related Links and Documents
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What is the exact localization of Human Dermal Lymphatic Endothelial Cells (HDLEC)? What medium should I use to culture them?
PromoCell’s HDLEC are isolated from skin (dermis). When isolated from juvenile donors (C-12216), the exact localization is foreskin. When derived from adult donors (C-12217), the localization depends on the type of surgery, e.g. breast or temple. You can find the information on the exact localization in the Certificate of Analysis.
Our HDLEC are tested to be positive for CD31, podoplanin, and ac-LDL uptake and are delivered in P2. The recommended culture medium is Endothelial Cell Growth Medium MV2 (C-22022). -
How should I use accutase for cell detachment?
Short protocol:
- Wash the cells with sterile PBS or HepesBSS
- Add undiluted accutase to the culture vessel (2 ml per 25 cm2)
- Incubate at room temperature for 5-15 min or at 37°C for faster detachment
- When the majority of the cells has detached, centrifuge the suspension and resuspend the pellet in fresh medium. In most cases no additional washes or neutralization steps are required.
Related Links and Documents
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What is the difference between DetachKit (C-41200) and DetachKit-2 (C-41202)?
Both Kits contain HepesBSS, trypsin/EDTA, and Trypsin Neutralizing Solution (TNS). In DetachKit (C-41200), the concentration of trypsin/EDTA is 0.04% / 0.03%. In DetachKit-2 (C-41202), it is reduced to 0.025% trypsin / 0.01% EDTA. HepesBSS and TNS concentrations are identical in both Kits.
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How does PromoCell characterize their MSC-AT (C-12977)?
Our hMSC-AT are characterized by their differentiation potential into chondrocytes, fat cells, and bone cells. In addition, we determine the presence of CD73, CD90 and CD105 expression as well as the absence of CD14, CD19, CD34, CD45 & HLA-DR expression by flow cytometry as proposed by the International Society for Cellular Therapy.
Related Links and Documents
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Do I have to use coated tissue culture vessels for culturing PromoCell Normal Human Cells?
It is not necessary to use coated flasks for (most of) our Normal Human Cells but it can be done. As coating with extracellular matrix proteins can affect cellular metabolism, it is recommended to use the same coating material for a complete set of experiments. Cells that need to be grown on coated dishes:
- Mesenchymal Stem Cells (C-12974/C-12971/C-12977) need Fibronectin- or Vitronectin-coating when grown in PromoCell MSC Growth Medium XF (C-28019) and when differentiated in MSC Neurogenic (C-28015), Adipogenic (C-28016), or Osteogenic (C-28013) Differentiation Media.
- Human monocyte-derived macrophages (C-12914/C-12916/C-12915/C-12917) must be seeded into Fibronectin- or Vitronectin-coated culture vessels in combination with PromoCell’s M1- and M2-Generation Media XF (C-28055, C-28056).
- For efficient induction of osteoblast mineralization with PromoCell’s Osteoblast Mineralization Medium (C-27020), the TC plates should be pre-coated with collagen type I.
Related Links and Documents
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At what passage do PromoCell test the cell type-specific markers?
Cell type-specific markers are usually determined one passage after thawing.
Example: After thawing, HUVEC are in P1. Markers (CD31 expression, Dil-Ac-LDL uptake) are tested in P2.
Please note: Cells that are frozen directly after isolation (blood cells) are tested directly after thawing with no further culturing step. -
What is the definition of “adult stem cells”?
“Adult stem cells” are stem cells isolated from postnatal tissues that have retained the capacity for cell renewal as well as for differentiation into multiple lineages (multipotency).
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What is the time frame for the differentiation of HWP? Can PromoCell provide a differentiation protocol?
Induction of differentiation:
– Grow the cells in Preadipocyte Growth Medium (C-27410) until they reach complete confluence; this roughly takes 1 week. Change medium 2-3x per week.
– Aspirate the Growth Medium; add Preadipocyte Differentiation Medium (C-27437) for 72 h
– Aspirate the Differentiation Medium, add Adipocyte Nutrition Medium (C-27438).
The cells will now start to accumulate droplets of lipids which can be visualized under the microscope. The process usually takes 1-2 weeks. Change medium 2-3x per week. It is possible to maintain the differentiated adipocytes in Nutrition Medium for up to 4 weeks. However, the cells tend to lyse and detach after about 3 weeks.Please note: We recommend to perform differentiation experiments at population doubling numbers lower than 4-5 (a maximum of 1 passage after thawing) in order to obtain a high differentiation level of the culture.
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The melanocytes from Promocell are primary cells, so how many passages can they go before losing activity?
PromoCell guarantee 15 population doublings for their NHEM. In terms of passages this corresponds to approx. 4-6 subcultivations. We can however not guarantee that the activity of all melanocyte genes remains unchanged during this time. Primary cells do gradually change their phenotype in vitro. Therefore, it is recommended to use the cells for the important experiments at low passages.
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Where does PromoCell source the heart tissue for HCM isolation?
In most cases, the tissue comes from patients who underwent heart transplantation. We obtain a part of the explanted (not the transplanted) heart to prepare the cardiac myocytes.
In other cases, the tissue comes from LVAD (= Left Ventricular Assist Device) surgery.Related Links and Documents
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