Technical Library

Recent Entries in Technical Library

• 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.

  Related Links and Documents

  • Renal Epithelial Cells
  • Schematic structure of a kidney
• 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

  Related Links and Documents

  • Cell Viability and Proliferation Kits
  • Kurs Zellviabilitäts-, Proliferations- und Toxizitätstests (German)
• 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.

  Related Links and Documents

  • HNEpC
  • DetachKit
• 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

  • SkMC
• 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.

  Related Links and Documents

  • Berthois et al.; Proc Natl Acad Sci U S A. 1986 Apr;83(8):2496-500
• 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

  • HCH
• 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.

  Related Links and Documents

  • Osteoblast Growth Medium
• 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.

   

  Related Links and Documents

  • Cell pellets
• 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.

• 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.

  Related Links and Documents

  • Osteoblast Media
  • Application Note: Osteoblast Differentiation and Mineralization
• 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.

  Related Links and Documents

  • Keratinocyte Growth Medium 2
  • NHEK
  • CaCl2 solution
• 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.

  Related Links and Documents

  • Human Umbilical Vein Endothelial Cells
• 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

  • Accutase Solution
• 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.

  Related Links and Documents

  • Freezing Medium Cryo-SFM
• 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

  • Blog article: Antibiotics in Cell Culture: Friend or Enemy?
• 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.

  Related Links and Documents

  • EXCiPACT™ GMP Certification
• 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. 

• 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.

  Related Links and Documents

  • Instruction Manual: Mesenchymal Stem Cells
  • Instruction Manual: MSC Media
  • Application Note: Neurogenic Differentiation and Analysis of MSC
• 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

  • Mesenchymal Stem Cells
  • MSC Growth Medium 2
• 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

  • Human White Preadipocytes
• 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

  • HDLEC
  • Endothelial Cell Growth Medium MV2
• 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

  • Accutase Solution
• 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

  • PromoCell Growth Medium Info Sheet
• 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.

  Related Links and Documents

  • hMSC
  • MSC Differentiation Media
  • Application Note: Osteogenic Differentiation and Analysis of MSC
• 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.

• 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.

• 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”.

  Related Links and Documents

  • Overview human primary cells
• 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).

  Related Links and Documents

  • HOB
• 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.

  Related Links and Documents

  • HCH
• 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.

  Related Links and Documents

  • HNEpC
• 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.

  Related Links and Documents

  • PromoCell DetachKit
  • PromoCell DetachKit 2
• 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.

   

  Related Links and Documents

  • Mesenchymal Stem Cell Culture
• 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

  • Application Note: Osteogenic Differentiation and Analysis of MSC
• 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.

  Related Links and Documents

  • Kumar et al.; Exp Eye Res . 2019 Dec;189:107860
• 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.

  Related Links and Documents

  • Human Uterine Fibroblasts
• 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

  • Application Note: Generation of moDCs
  • Dendritic Cell Culture
  • PromoCell DC Generation Media
• 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

  • Human White Preadipocytes and related media
• 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.

• 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%.

  Related Links and Documents

  • HDMEC
• 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.

  Related Links and Documents

  • Trypsin Neutralizing Solution (TNS)
• 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

  • DetachKit
• 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.

  Related Links and Documents

  • Product Manual: hMSC
  • Application Note: Adipogenic Differentiation and Analysis of MSC
  • Application Note: Osteogenic Differentiation and Mineralization of MSC
  • Application Note: Chondrogenic Differentiation and Analysis of MSC
  • Application Note: Neurogenic Differentiation and Analysis of MSC
• 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”).

  Related Links and Documents

  • Human Stem & Blood Cell Culture
• 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.

• 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.

  Related Links and Documents

  • HOB
• 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.

  Related Links and Documents

  • Chondrocyte Growth Medium
• 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

  • HBEpC
• 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

  • Endothelial Cell Culture
  • Howell et al.; Mol Membr Biol. Nov-Dec 2004;21(6):413-21
• 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.

  Related Links and Documents

  • Application Note: Osteogenic Differentiation and Analysis of MSC
  • human Mesenchymal Stem Cells
  • Mesenchymal Stem Cell Osteogenic Differentiation Medium
• 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.

   

   

  Related Links and Documents

  • NHEK
  • NHEK in GM3
• 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.

  Related Links and Documents

  • App Note: Air-Liquid Interface Culture of Nasal and Tracheal Airway Epithelial Cells
  • App Note: Air-Liquid Interface Culture System for Standardized Respiratory Research (HBEpC)
  • Air liquid interface medium
  • Products for epithelial cell culture
• What could be the reason for a lower yield when using M2 Macrophage Generation Medium compared to M1 Macrophage Generation Medium for a culture started with the same monocytes?

  For the M2 Macrophage Generation Medium, it is extremely important that the shelf life of 2 weeks (after addition of the cytokines) is not exceeded; the yield will quickly decrease thereafter. It is best to use the M2 medium as fresh as possible to avoid discrepancies between M1 and M2 yield.

  Related Links and Documents

  • Application Note: Differentiation of M1- or M2-Macrophages from PBMC/Monocytes
  • Macrophage Generation Media DXF
• Can PromoCell recommend a medium to co-culture endothelial cells and fibroblasts?

  Some of our customers have successfully used PromoCell Endothelial Cell Growth Medium MV2 (C-22022) when co-culturing Human Coronary Artery Endothelial Cells (HCAEC; C-12221) and Normal Human Dermal Fibroblasts (NHDF; C-12300).

  Related Links and Documents

  • Endothelial Cell Growth Medium MV2
• What are the advantages of using PromoCell DC Generation Medium XF for dendritic cell generation?

  Our DC Generation Medium XF (C-28052) has a xeno-free formulation. It provides a complete media system (ready-to-use, all cytokines included) and shows efficient and reproducible in vitro maturation of moDCs from freshly isolated peripheral blood monocytes.

  Related Links and Documents

  • Dendritic Cell Generation Medium XF
• How does PromoCell ensure that there is no fibroblast contamination in the pericytes? Are the fibroblasts removed by separation with anti-CD90 labeled magnetic beads?

  No, we don’t perform CD90 immunomagnetic separation with our pericytes. We can however exclude fibroblast contamination as follows:
  1) The presence of fibroblasts in our pericyte cultures would be detectable shortly after cell isolation, as pericytes need up to 2 weeks before they start proliferating. Fibroblasts on the other hand would proliferate immediately and overgrow the culture.
  2) Characterization of the isolated pericytes during QC includes flow cytometry of CD146. Pericytes express CD146 whereas placental fibroblasts don’t.

  Related Links and Documents

  • Human Pericytes from Placenta
• My cells didn’t detach during subculture. What could be the reason?

  Please find attached a trouble shooting guide to identify possible reasons for poor detachment during subculture.

  Related Links and Documents

  • Trouble shooting guide: Slow detachment
• Why is the growth rate of PromoCell’s HDMEC significantly reduced when I remove hydrocortisone from the Endothelial Cell Growth Medium MV Kit (C-22120)?

  Epidermal Growth Factor (EGF) and hydrocortisone have been reported to have a synergistic effect on the growth of microvascular endothelial cells. Accordingly, if you remove the hydrocortisone, the proliferation of HDMEC is clearly affected.

  Related Links and Documents

  • Endothelial Cell Growth Medium MV
• Should I warm the trypsin prior to use?

  We recommend to use the trypsin as well as the other detach solutions at room temperature to avoid overtrypsinization and irreversible cellular damage.

  Related Links and Documents

  • DetachKit
• 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.

  Related Links and Documents

  • DetachKit
• 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

  • hMSC-AT
• 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-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-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

  • Mesenchymal Stem Cell Culture
  • Macrophage Cell Culture
  • Osteoblast Cell Culture
• 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).

  Related Links and Documents

  • Human Stem & Blood Cell Culture
• 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).

  Related Links and Documents

  • Osteoblast Media
• 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 Cryo-SFM (C-29910) which is a serum-free freezing medium that prevents the possibility that any serum is introduced into your serum-free cell culture system. It is also recommended to freeze the cells at an early passage.

  Related Links and Documents

  • Melanocyte Cell Culture
  • Freezing Medium Cryo-SFM
• 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

  • HNEpC
• 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.

  Related Links and Documents

  • Endothelial Cell Culture
  • Ando et al.; Am J Physio l. 1999 May;276(5):H1755-68
  • Lu et al.; Stem Cells Dev . 2004 Oct;13(5):521-7
  • Azuma et al.; Biochem Biophys Res Commun . 2009 Mar 13;380(3):620-6
  • Frid et al.; Circ Res . 2002 Jun 14;90(11):1189-96
  • Kovacic et al.; J Am Coll Cardiol . 2019 Jan 22;73(2):190-209
  • Jones et al.; Am J Respir Cell Mol Biol . 1999 Apr;20(4):582-94
  • Ma et al.; Front Cell Dev Biol . 2020 Apr 21;8:260
  • Kocher and Madri; In Vitro Cell Dev Biol . 1989 May;25(5):424-34
  • Amberger et al.; FEBS Lett . 1991 Aug 5;287(1-2):223-5
• Which human AB serum does PromoCell recommend to generate M0 macrophages?

  We recommend to use Human Serum AB „off-the-clot”.

  Related Links and Documents

  • Macrophage Base Medium DXF
  • Application Note: Convenient large-scale differentiation
• 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).

   

  Related Links and Documents

  • Macrophage Detachment Solution
  • Campuzano et al.; J Immunol . 2020 Jun 15;204(12):3296
• 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

  • AppNote: Generation of monocyte derived Dendritic Cells (moDCs)
  • Dendritic Cell Generation Medium
  • Human CD14+ Monocytes
• After detaching the macrophages for flow cytometric analysis, I noticed that the washing and centrifugation steps take a long time. Can I shorten the spin time to 10 minutes at 350 x g?

  Spinning the cells for 15 min at 350 x g has been proven and tested by PromoCell Research & Development. The QC department uses these settings during testing. Any lower centrifugation value (g-force and/or time) will lead to significant cell loss by means of non-sedimented, but intact, macrophages.

  Related Links and Documents

  • Differentiation of M1- or M2-Macrophages from PBMC/Monocytes
  • Macrophage Generation Media DXF
  • Macrophage Detachment Solution DXF
• We are culturing human coronary artery smooth muscle cells (HCASMC) from your company. Are these cells obtained from large arteries like LAD or left Circumflex or from small branches of LAD etc. ?

  Our HCASMC (C-12221) are isolated from the large arteries, i.e. from

  • Right coronary artery
  • Left main coronary artery
  • Circumflex coronary artery and
  • Left anterior descending coronary artery.

  Related Links and Documents

  • HCASMC
• How long does it take to differentiate monocytes into mature DCs using PromoCell DC Generation Medium/DC Generation Medium XF?

  It takes 7-8 days to generate fully mature myeloid dendritic cells.

  Related Links and Documents

  • Application Note: Generation of moDCs
  • Dendritic Cell Generation Medium XF
  • Dendritic Cell Generation Medium
• What is the exact localization of PromoCell’s Human Pericytes? How many doublings can they perform in culture?

  Our hPC-PL (C-12980) are isolated from microvessels of the human placenta, from the chorionic villi.
  The number of populations doublings is not determined for each individual cell lot, but in our experience, they can be grown for at least 15 population doublings.

  Related Links and Documents

  • hPC-PL
• My Normal Human Cells didn’t attach after thawing. What could be the reason?

  Poor attachment after thawing can be a result of inappropriate freezing, storing or thawing the cells as well as from inadequate culture conditions (medium, incubator). The attached trouble shooting guide should help you to identify the possible reasons.

  Related Links and Documents

  • Trouble shooting guide: Poor attachment
  • Thawing of primary cells
  • Best Practices in Cell Culture
• Is it possible to split differentiated adipocytes in order to perform more experiments?

  Generally, you can split differentiated adipocytes. But these cells lose their ability to proliferate after differentiation and you can’t expand the culture any more. Therefore it is recommended to plate the cells into the needed vessels (e.g. multiwell plates) prior to induction of differentiation so that trypsinization isn’t necessary.

  Related Links and Documents

  • HWP
• What is the concentration of the trypsin inhibitor in TNS?

  Our TNS solution contains 0.05% (w/v) trypsin inhibitor from soy bean in HepesBSS/0.1% BSA.

  Related Links and Documents

  • TNS
• What is the shelf life of the PromoCell DetachKit/DetachKit-2?

  The three components of DetachKit and DetachKit-2 (HepesBSS, Trypsin/EDTA, TNS) are stable for 1 year from the date of manufacture when stored at -20°C. Once thawed for usage, they should be stored at 4-8°C and can be used for 6 weeks. Please avoid repeated freeze/thaw cycles.

  Related Links and Documents

  • DetachKit
  • DetachKit
• I would like to know the number of population doublings for PromoCell mononuclear cells (C-12907/C-12901) when grown in Mononuclear Cell Medium (C-28030).

  Mononuclear cells are mostly used in immunology, infection biology, hematology and cancer research to study subpopulations of blood cells.
  Our Mononuclear Cell Medium (C-28030) is intended for short-term maintenance (up to 48 hrs) of the thawed hMNC before you proceed with your experiments. The number of PDs will depend on the subsequent cell culture conditions and is not determined by PromoCell.
  Please note: Depending on the conditions, the ratio of the subpopulations will gradually change, as the different blood cell types behave in different manners. Researchers normally start soon after thawing to either select the cell type of their interest (e.g. hematopoietic cells, endothelial progenitor cells) or perform experiments with all populations of hMNCs (e.g. to study effects on toxicity, viability or metabolism).

  Related Links and Documents

  • hMNC
  • Mononuclear Cell Medium
• How can I calculate the population doubling time?

  The population doubling time or generation time (t_g) is usually calculated during the logarithmic phase of growth. It specifies the time (t) in hours needed by the culture to double its cell number.
  
  t_g = t / n

  n: number of population doublings

• What is the precise localization for a) HNEpC, b) HTEpC, c) HBEpC, and d) HSAEpC?

  a) HNEpC (Human Nasal Epithelial Cells) are isolated from nasal mucosa
  b) HTEpC (Human Tracheal Epithelial Cells) from the surface epithelium of trachea
  c) HBEpC from the surface epithelium of bronchie, and
  d) HSAEpC (Human Small Airway Epithelial Cells) from the distal portion of the respiratory tract in the 1 mm bronchiole area
  (comprising the cells from bronchioli and alveoli).

  Related Links and Documents

  • Human Small Airway Epithelial Cells
• What is the difference between “primary cells” and “normal cells”?

  Freshly isolated cells that are plated in a tissue culture vessel for the first time are named primary cells or primary culture (corresponding to P0). As soon as they have been subcultured, they should correctly be termed normal cells (> P1).

• 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-27436) 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.

  Related Links and Documents

  • HWP
  • Preadipocyte/Adipocyte Media
  • HWP and related media
• 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.

  Related Links and Documents

  • Melanocyte Cell Culture
• Where does PromoCell source the heart tissue for HCM isolation?

  The tissue comes from patients who underwent heart transplantation. We obtain a part of the explanted (not the transplanted) heart to prepare cardiac myocytes.

  Related Links and Documents

  • Human Cardiac Myocytes
• I received 3 DetachKits yesterday. Some of the tubes have different colors, although they all come from the same batch.

  The components of the PromoCell DetachKit may occasionally arrive with a non-uniform color appearance. This phenomenon is known to PromoCell’s Quality Assurance Dept.
  It is reversible and has no influence on the quality of the product.

  Related Links and Documents

  • PromoCello DetachKit
• Will the NHEM.f (C-12400) and NHEM.f M2 (C-12402) also grow in PromoCell Melanocyte Growth Medium M3?

  Yes, NHEM.f and NHEM.f M2 also grow in our optimized Melanocyte Growth Medium M3 (C24310) and can achieve > 15 population doublings.

  Related Links and Documents

  • Melanocyte Cell Culture
• Why is the actual cell count in the vial at PromoCell sometimes significantly higher than the 500,000 cells stated on the website?

  We guarantee > 500,000 viable cells per vial. To do this, we need to freeze more than 500,000 cells, as we do not yet know the viability after thawing at the time of freezing.
  For technical/organizational reasons, the initial cell number may also vary from lot to lot, so 2 lots with the same viability may not necessarily contain the same number of cells.

  Related Links and Documents

  • Human primary cell culture
• Has PromoCell done any tests with MSC Adipogenic Differentiation Medium 2 without fibronectin? Is fibronectin coating really necessary?

  The MSCs attach even without fibronectin coating, but the risk of detachment during differentiation is much higher. We therefore strongly recommend using fibronectin-coated TC vessels.

  Related Links and Documents

  • Human Mesenchymal Stem Cells
  • Mesenchymal Stem Cell Adipogenic Differentiation Medium 2