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
290 Entries on 6 Pages
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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 phenol red-free basal media for cell culture?
It has been shown that phenol red has estrogenic properties. Phenol red-free media are therefore generally used in studies evaluating steroid hormone action in cultured, estrogen-responsive cells (Berthois et al., 1986). Furthermore, phenol red can also interfere with some analytical methods like photometric analyses.
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What chondrocyte structures are stained using Alcian Blue? Can I use this staining method for chondrocytes in monolayer cultures?
Alcian Blue stains the extracellular matrix of chondrocytes, e.g. cartilage-specific aggrecan and other glycosaminoglycans. To our knowledge, chondrocytes only express aggrecans when grown in 3-D culture and not in 2-D culture.
To detect cartilage specific markers in monolayer culture, it is recommended to perform immunofluorescence detection of collagen type II.Related Links and Documents
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Does PromoCell recommend growing their osteoblasts on type I collagen?
PromoCell generally culture their HOB on uncoated tissue culture dishes. It is possible however to grow them on collagen type I- or fibronectin-coated dishes as well. Please note: The type of extracellular matrix used may influence the expression of certain genes (e.g. integrins) and thereby affect cellular metabolism. Therefore, we recommend to always use the same type of coating matrix for a whole set of experiments.
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How do I best isolate RNA from PromoCell cell pellets (C-14**)?
There are two options for isolating RNA from cells stored in RNAlater Solution:
1) The solution is removed from the cells prior to extraction by centrifugation at 5,000 x g for 10 minutes at 4°C.
Note: Because of the density of RNAlater® solution, greater centrifugal forces are required to spin down the cells.2) If no pellet is visible after centrifugation, RNA can also be purified directly from the RNAlater® solution. This can be done by adding 2 ml of 10x lysis buffer, and proceeding normally.
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What experience do I need to grow PromoCell Normal Human Cells, Adult Stem and Blood Cells?
You need to have experience working under sterile conditions and under a laminar flow hood. It is of advantage to have experience with other cell types and/or cell lines. If you are a beginner in cell culture and would like to establish a cell culture lab, we will assist you in working with PromoCell Normal Human Cells.
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How long is it before the osteoblasts produce mineralized nodules?
For mineralization assays, HOB are cultured in Osteoblast Mineralization Medium (C-27020). Mineralization can be detected after approximately 3 weeks by incorporation of Ca-45, or it can be visualized by von Kossa or Alizarin Red staining for calcium.
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What calcium concentration is needed to induce differentiation in cultured keratinocytes?
The optimal calcium concentration for both proliferation and differentiation of keratinocytes depends on the species and also on the media formulation. To keep primary human keratinocytes in the proliferative status, concentrations between 0.03 and 0.15 mM (PromoCell Keratinocyte Growth Medium 2: 0.06 mM) are generally used. Increasing the calcium above 1 mM will induce terminal differentiation and lead to the loss of proliferative activity.
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Will precoating of dishes have any adverse impact on the HUVEC?
Precoating of culture vessels with ECM proteins does not have adverse effects on the cells but has been reported to influence the cellular expression pattern. It is therefore recommended to use the same culture conditions, e.g. fibronectin-, collagen-, or gelatin-coating for a whole set of experiments to be able to compare the results.
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Can I defrost Accutase Solution, prepare aliquots and refreeze them?
Yes, Accutase Solution can be defrosted, aliquoted, and then refrozen.
Defrosting: Accutase should be defrosted overnight in the refrigerator or placed in a tub of cold tap water. Do not defrost in a 37°C water bath.
Stability: Once thawed, it is stable for at least 2 months in the refrigerator if stored promptly after use.Related Links and Documents
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Is PromoCell’s Cryo-SFM produced under GMP standard?
No, our Freezing Medium Cryo-SFM (C-29910) is not produced under GMP standard. It is for in vitro research use only and not appoved for diagnostic or therapeutic procedures.
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What are the disadvantages of the prophylactic use of antibiotics in cell culture?
The use of antibiotics creates a false sense of security and allows users to develop poor aseptic techniques. This leads to low-level contamination with partially resistant bacteria occurring but being overlooked for a time. This then leads to cells with undetected contamination being cultured for extended periods of time, increasing the risk that contamination will spread throughout the laboratory and eventually antibiotic-resistant strains of bacteria may develop.
Mycoplasma infections can also occur more easily, as they are often introduced along with contaminants such as bacteria and fungi.
Last but not least, antibiotics are known to have negative effects on the metabolism of eukaryotic cells – more details on this topic can be found in our blog article “Antibiotics in cell culture: friend or enemy”.Related Links and Documents
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Are your cells isolated under GMP?
Our cells are not manufactured according to GMP guidelines and are intended for in vitro use only.
Our EXCiPACT™ GMP certification only applies to the processes related to the media.
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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 Prox-1 and are delivered in P2. The recommended culture medium is Endothelial Cell Growth Medium MV2 (C-22022).Related Links and Documents
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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.
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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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How many osteoblasts will be in the flask when I purchase a proliferating culture (C-12760)?
If you purchase a proliferating culture of human osteoblasts (C-12760), there will be > 500,000 cells in the TC-flask. Once the culture is subconfluent, you will count between 750,000 – 1.1 million cells/T25 (depending on the cell lot).
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Typically once seeded, how long does it take to grow the chondrocytes to subconfluence? How many doublings will they undergo per passage?
It usually takes 5 to 8 days to grow our Normal Human Chondrocytes (C-12710) to subconfluency. The number of doublings (PDs) they undergo can be calculated from the number of seeded cells and the cell yield at subconfluency. Generally, when HCH are plated with 10,000 cells/cm² they perform between 1.5 and 2 doublings per passage.
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Is it necessary to cultivate Nasal Epithelial Cells on coated culture flasks?
No, it is not necessary to use coated flasks, therefore we don´t recommend their usage in the Instruction Manual. However, for special applications, some of our customers use collagen-coated dishes.
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The components of my DetachKit show different colors. Is this normal or does it affect their quality?
The components of the PromoCell DetachKit may arrive on occasion with a non-uniform color appearance. This phenomenon is known by PromoCell’s Quality Assurance. It is reversible and does not influence the quality of the product.
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I am performing MSC chondrogenic, adipogenic, and osteogenic differentiation and am staining the cells. The protocol says to use Saccomanno Fixation Solution. Is there an alternative method to fix the cells because I don’t have Saccomanno Fixation Solution?
Yes, you can use 4.5% neutral buffered formalin. Paraformaldehyde should work as well.
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Why is PBS added in the last step of the Alizarin Red staining protocol (“Osteogenic differentiation and analysis of MSC”)? Should the PBS be aspirated before analysis?
The PBS buffering enhances the Alizarin Red staining (precipitation of the dye) and makes it more intense.
Leave the PBS on the cells after staining/washing and analyze the sample immediately, as the dye may bleed upon prolonged storage without embedding.Related Links and Documents
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What is the shelf life of primary cells cryopreserved in liquid nitrogen?
PromoCell cells are frozen down in the gas phase of liquid nitrogen (computer-controlled freezing machine) and then stored in the liquid phase of LN2.
The cryopreservation in LN2 is an acknowledged method for long-term storage of primary cells and stem cells. When stored in liquid nitrogen, the cells can be maintained for a period of > 10 years without affecting viability.
For example, Kumar et al. showed that adipose-derived stem cells stored in LN2 for about 12 years still retained their regenerative potential, stem cell property, viability as well as differentiation ability.
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Do all PromoCell HUF come from the myometrium or does the uterine location vary by lot?
All our Human Uterine Fibroblasts (HUF) are prepared from the myometrium.
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Which DC medium does PromoCell recommend for freshly isolated MNC or monocytes, which one for cryopreserved monocytes?
The PromoCell DC Generation Media have been developed for the easy and efficient generation of immature as well as fully mature myeloid dendritic cells from peripheral blood monocytes.
For freshly isolated mononuclear cells (MNC) and monocytes we recommend our DC Generation Medium XF (C-28052), for cryopreserved monocytes our DC Generation Medium (C-28050). When using DC Generation Medium (C-28050) with fresh MNC, the Monocyte Attachment Medium (C-28051) is needed in a first step for efficient adherence of the monocyte fraction.
Please see Instruction Manual, Application Note and the graph below for further details.Related Links and Documents
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What are the key points relating to proliferation, differentiation and culturing of HWP?
PromoCell’s Normal Human White Preadipocytes (HWP) are isolated from adult subcutaneous or visceral adipose tissue from different locations. The cells are frozen in our serum-free freezing medium (Cryo-SFM) at the end of passage 1 (= secondary culture). A randomly selected vial is then used for quality control, which includes determination of growth characteristics, control of morphology, and tests for differentiation capacity into mature adipocytes.
The recommended seeding density of preadipocytes after thawing/trypsinization is 5,000 cells/cm2; cells should be trypsinized before reaching 90% confluence. Population doubling times are usually between 20-50 hrs (10 population doublings guaranteed). Using a 1:4 split ratio, you can perform ∼4-5 passages with the cells.
Preadipocyte Growth Medium (C-27417) is used to propagate the cells. To induce differentiation of preadipocytes into adipocytes, cells are grown in PromoCell’s Preadipocyte Growth Medium until they reach 100% confluency. Cells are then cultured in Preadipocyte Differentiation Medium (C-27437) for 72 h, followed by 10-14 days in Adipocyte Nutrition Medium (C-27439). During this time the cells start to accumulate fat droplets which can be visualized under the microscope.
We recommend performing differentiation experiments at population doubling numbers lower than 4-5, in order to reach a high differentiation level of the culture.Related Links and Documents
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Why is the serum content of PromoCell’s specialized media so low?
FCS is a natural product consisting of many different components like salts, hormones, vitamins, trace elements, proteins, and enzymes. There can be large lot-to-lot variations between different serum batches regarding the concentrations of growth-promoting factors. The higher the serum content in a culture medium, the higher the impact of the variations on the cell culture system. For this reason, PromoCell has developed several serum-reduced and serum-free media where part of the serum has been replaced by more defined factors like cytokines, hormones or vitamins.Media with reduced serum concentrations have the benefit that they produce more standardized culture conditions over a long time span.
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Does PromoCell determine the percentage of HDLEC in its HDMEC lots?
No, we don’t determine the ratio of HDLEC and HDBEC in our HDMEC lots. From our experience, the percentage of HDLEC is highly lot-dependent and can vary between 5 and 60%.
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Can I use the Growth Medium instead of TNS to stop the trypsin when splitting the cells?
A concentration > 10% FBS is needed to completely inactivate the trypsin. As most of PromoCell’s growth media are serum-reduced or serum-free, the use of a trypsin inhibitor like TNS is highly recommended.
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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”. -
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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