Technical Library

Recent Entries in Technical Library

• 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 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 20-40 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 our Cryo-SFM (C-29910). 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
• I would like to know if the Lymphocyte Separation Medium (C-44010) is endotoxin tested.

  Yes, our Lymphocyte Separation Medium 1077 is endotoxin-tested. The specification for the product is < 10EU/ml.

  Related Links and Documents

  • Lymphocyte Separation Medium
• Is the PromoCell Keratinocyte Growth Medium 2 suitable for growth of primary mouse keratinocytes?

  Yes, primary mouse keratinocytes grow well in this media when you reduce the CaCl₂ concentration to 0.025 mM – 0.05 mM CaCl₂ (optimal Ca concentration for primary human keratinocytes is 0.06 mM).

  Related Links and Documents

  • Keratinocyte Growth Medium 2
• When do I need PromoCell’s Monocyte Attachment Medium (C-28051)?

  The Monocyte Attachment Medium allows for the efficient adherence selection (within 1 hr) of monocytes from freshly isolated mononuclear cells while maintaining optimal cell health. The Time-consuming and costly immunomagnetic purification of monocytes prior to DC generation is not necessary and can be skipped when using this medium.
  .

  Related Links and Documents

  • Instruction Manual: DC Media
  • Application Note: Generation of moDCs
  • Use of DC Attachment Medium
• How long does it take to detach primary cells with accutase (C-41310)?

  Most primary cells detach within 5-10 min at 37°C. Inactivation isn’t required but we recommend to centrifuge the cell suspension to remove accutase and EDTA before replating the cells.

  Related Links and Documents

  • Accutase Solution
• Is it possible to transfect PromoCell Normal Human Cells?

  Yes, it is possible to transfect our normal human cells. In general, primary and normal cells are much harder to transfect than immortalized cell lines. Apart from the cell type, successful transfection also depends on the culture’s age and density at transfection, the vector used, the purity of the nucleic acids, the composition of the transfection medium, and the experimental conditions.

• What’s the stability of HPC Expansion Medium XF (C-28021) after addition of Cytokine Mix E (C-39890)?

  The medium supplemented with Cytokine Mix E is stable for 2 weeks if stored protected from light at 2-8°C.

  Related Links and Documents

  • Hematopoietic Progenitor Expansion Medium XF
• Is it necessary to heat inactivate FCS for use with Normal Human Cells?

  The practice of heat inactivation was originally developed when only serum from adult animals was available. Adult serum contains high serum complement which may destroy cells under certain conditions. Heating serum (30 min, 56°C) is intended to inactivate the complement. Today, serum is often heat-inactivated without any evidence of beneficial effect. When using FCS (fetal calf serum), heat inactivation is not necessary for most cell lines or cell types. PromoCell does not use heat-inactivated FCS for the preparation of their growth media.

• Can I use my own culture medium or other commercially available media for culturing PromoCell Normal Human Cells?

  Our Normal Human Cells have been cultured and tested in our growth media and have adapted to these conditions. Using other media may yield unsatisfactory results due to suboptimal supplies of nutrients and growth factors. PromoCell can only guarantee good cell growth (as stated in the Certificate of Analysis), when the cells are grown in the recommended media.

• Does the differentiation capacity of Human Mesenchymal Stem Cells change over time? When should I best induce differentiation?

  We have tested the differentiation capacity of our hMSC into adipocytes, chondrocytes and osteoblasts over time and still see good differentiation rates after 10 population doublings, i.e., at passage 5. However, the differentiation potential declines with ongoing population doublings. To obtain optimal differentiation rates, experiments should be performed as early in culture as possible.

  Related Links and Documents

  • Mesenchymal Stem Cell Culture
• How many cells do the cell pellets contain?

  PromoCell cell pellets are made from > 1 million cells and are dissolved in 200 µl RNAlater.

• What are the characteristic markers of HDLEC (C-12216, C-12217) and HDBEC (C-12211, C-12225)?
  • HDLEC and HDBEC both express the typical endothelial cell marker CD31 (= PECAM-1).
  • HDLEC cultures are additionally tested positive for podoplanin, a transmembrane glycoprotein involved in lymphatic vessel formation, whereas HDBECs are podoplanin-negative.

  Related Links and Documents

  • Microvascular Endothelial Cells
• How often should I change the growth medium when culturing PromoCell Normal Human Cells?

  Generally, the medium should be changed every 2-3 days.

  Please note: Following thawing, the first medium change should be performed after 16-24 hours to prevent cell damage due to residual freezing medium.

• Is everything in the PromoCell Skeletal Muscle Cell Differentiation Medium (C-23061) defined?

  Yes, our Skeletal Muscle Cell Differentiation Medium is completely defined. The SupplementMix (C-39366) consists of recombinant human insulin.

  Related Links and Documents

  • Skeletal Muscle Differentiation Medium
• How can I be sure that the cells in my melanocyte culture are melanocytes? Have I understood it correctly that the medium is selective for melanocytes?

  After isolation and purification of our primary human melanocytes, the cells are checked during quality control whether they show a typical morphology and whether they express the marker Mel-5. Mel-5 is a 75 kDa glycoprotein usually expressed by normal melanocytes.
  Our Melanocyte Growth Medium (C-24010) has been developed to promote melanocyte growth in vitro. It does not however, completely block the growth of other cells (such as: NHEK or NHDF). Therefore, it is important to have a pure melanocyte culture from the very beginning.
  In contrast, the Melanocyte Growth Medium M2 (C-24300) and Medium M3 (C-24310) are much more selective and repress the growth of contaminating cells much better.

  Related Links and Documents

  • Melanocyte Cell Culture
• For how long can the cardiac myocytes (C-12810) be grown in culture?

  PromoCell guarantees 15 population doublings (PDs) if the HCM are grown in Myocyte Growth Medium. Depending on the cell lot and the culture conditions, the cells can be maintained in culture for > 6-8 passages corresponding to a period of 1-2 months.

  Related Links and Documents

  • Human Cardiac Myocytes
  • Myocyte Growth Medium
• Did PromoCell try plating the cryo-macrophages in 96-well plates?

  We did not plate the cryo-macrophages in 96-well plates. However, we heard from other customers that they have successfully used our macrophages in this kind of plate.

  Related Links and Documents

  • Macrophage Cell Culture
• Will my NHEK (isolated in PromoCell Keratinocyte Growth Medium 2) grow in PromoCell Keratinocyte Growth Medium 3?

  Yes, the NHEK-GM2 (C-12001, C-12003, C-12005, C-12006) also grow in PromoCell Keratinocyte Growth Medium 3 (C-20021). Using the protocol with the fixed intervals, they grow slightly faster than in Keratinocyte GM2 and proliferate for > 15 PDs.
  Conversely, NHEK-GM3 (C-12011, C-12013, C-12015, C-12016) also grow in the existing Keratinocyte Growth Medium 2 (C-20011). When using the classical subcultivation protocol (density > 70% – 90%), they grow slightly slower compared to Keratinocyte GM3, but also reach > 15 PDs.

  Related Links and Documents

  • Keratinocyte Cell Culture
• For which cytokeratin are the keratinocytes tested?

  PromoCell is using a pan-cytokeratin antibody rather than a specific type of cytokeratin for the quality control of our keratinocytes.

  Related Links and Documents

  • Normal Human Epidermal Keratinocytes
  • Normal Human Epidermal Keratinocytes GM3
• Can I thaw a vial of HBEpC and directly seed it on transwells for my ALI-experiment?
  Yes, but remember to thaw and seed the cells in our growth factor containing Airway Epithelial Cell Growth Medium as the cells need to expand and proliferate for some days.

  Always use a seeding density of 150.000 cells/cm², even if you plate the cells on transwells directly after thawing. Do not forget to coat the inserts with 30 µg/ml Collagen Type I solution (e.g. Corning Inc^®., product number 354236) before you seed the cells.

  Related Links and Documents
• Can I use the 3D Tumorsphere Medium XF for mouse cell lines?

  Yes, we have received a customer feedback that our 3D Tumorsphere Medium XF (C-28070) has been successfully used for tumorsphere formation of mouse cell lines.

  Related Links and Documents

  • Tumorsphere Culture of Cancer Stem Cells (CSC) with the PromoCell 3D Tumorsphere Medium XF
  • 3D Tumorsphere Medium XF
• Looking at the detachment protocol of PromoCell’s Macrophage Detachment Solution, I observed that we need to add HSA to the PBS. Can I use FBS instead? Also, why are we adding 2 mM EDTA to the PBS?
  • FBS (or BSA) can be substituted for HSA, however we do not recommend this as the FBS will lead to unpreferable immunologic stimulation of human macrophages.

  • The addition of EDTA to the PBS is to further augment the “anti-clumping” activity of the Ca²⁺/Mg²⁺ free PBS.

  Related Links and Documents

  • Macrophage Detachment Solution
• What is the difference between DC Generation Medium Ready-to-use (C-28050) and DC Base Medium (C-28053)?
  • DC Base Medium (C-28053) consists of 250 ml Basal Medium + SupplementMix. It does not include cytokines and must therefore be supplemented according to the user’s individual needs.

  • DC Generation Medium (C-28050) is ready-to-use and consists of 250 ml Basal Medium + SupplementMix + cytokines.

  Related Links and Documents

  • Instruction Manual: DC Media
  • Application Note: Generation of moDCs
• Do the PromoCell media contain L-glutamine?

  Yes, the specialized PromoCell media already contain the optimal amount of L-glutamine. Please don’t add extra L-glutamine as this can be toxic for the cells.

• What’s the difference between trypsin and accutase?

  Both, trypsin and accutase represent mixtures of different proteolytic enzymes. Trypsin is prepared from porcine pancreas, accutase from invertebrates. Accutase can replace trypsin for the detachment and dissociation of anchorage-dependent cells from surfaces and can also be used on suspension cells to reduce clumping in preparation for counting. The advantages of accutase over the traditional trypsin treatment are that it is more gentle and less damaging to cells (leading to increased viability) and does not contain any mammalian or bacterially derived proteins. Accutase is more thermolabile than trypsin and usually doesn’t require an inactivation step.

  Related Links and Documents

  • Trypsin
  • Accutase
• Why are most of the soluble receptors not produced in E. coli?

  Soluble receptors are post-translationally modified and generally contain disulfide bonds and glycosylation sites. If produced in E. coli the proteins don’t show any biological activity.

  Related Links and Documents

  • Cytokines and Growth Factors
• What is the DMSO concentration in Cryo-SFM?

  The DMSO concentration in our Cryo-SFM is 10%.

  Related Links and Documents

  • Freezing Medium Cryo-SFM
• How do mesenchymal stem cells from different tissues differ in terms of their biological function?

  The human MSC derived from bone marrow, adipose tissue, and umbilical cord matrix are from different origins, but with comparable biological properties and function. Depending on the tissue of origin, they may have a higher preference for differentiation into one particular cell type and a lower preference for another one, but they all still retain the differentiation potential for the mesenchymal lineage.

  • MSC-BM: very good differentiation into bone cells, chondrocytes & fat cells
  • MSC-UC: very good differentiation into chondrocytes; but weaker potential into fat/bone cells
  • MSC-AT: very good differentiation into fat cells

  Related Links and Documents

  • Mesenchymal Stem Cell Culture
• Which plates does PromoCell recommend as 96-well U bottom suspension plates for chondrogenic differentiation of Human Mesenchymal Stem Cells?

  For chrondrogenic differentiation it is important that the cells do not adhere to the wells. During differentiation, the cells form spheroids which float in the medium. Therefore, there are no special requirements for the wells as long as they are U bottom shaped and suitable for suspension culture.

  Related Links and Documents

  • hMSC
  • Chondrogenic Differentiation and Analysis of MSC
• At what passage do PromoCell supply the cell pellets?

  The majority of PromoCell cell pellets (C-14**) are prepared 1 passage after thawing the cryopreserved cells.

  Examples:
  HUVEC and HUAEC correspond to P1 after thawing; therefore the pellets are frozen in P2.
  SMC or keratinocytes or epithelial cells correspond to P2 after thawing; therefore the pellets are frozen in P3.
  In contrast, our blood cells are cryopreserved directly after cell isolation. These pellets are prepared after thawing the cryovials with no further cultivation step.

• From what tissue do PromoCell isolate their HUtMEC (C-12295)?

  Our HUtMEC are isolated from the middle layer of the uterine wall (myometrium). The tissue donors were not pre-treated with hormones.

  Related Links and Documents

  • HUtMEC
• What is the composition of the Osteoblast Supplement (C-39615)?

  Our Osteoblast Supplement consists of FCS (10 % [v/v] final concentration) which is specifically tested to support optimal growth of Normal Human Osteoblasts.

  Related Links and Documents

  • Osteoblast SupplementMix
• What would be the likely impact of using PromoCell Fibroblast Growth Medium (C-23010) instead of DMEM + 10% FCS for the growth of juvenile fibroblasts (C-12300)?

  PromoCell Fibroblast Growth Medium is serum-free and therefore not subjected to the lot-to-lot variations observed with DMEM/10% FCS. Our Fibroblast Growth Medium therefore allows for much more standardized cell culture conditions.

  Related Links and Documents

  • Fibroblast Growth Medium
  • NHDF
• Can PromoCell supply melanocytes from asian donors?

  The majority of our skin tissue donors are caucasians. But occasionally we also get skin biopsies from asian and black donors.
  Please contact our Technical Customer Support if you need cells from a particular phototype or origin. They will check our inventory and send you a list of available cell lots.

  Related Links and Documents

  • Melanocyte cell culture
• How many bottles of medium will I need to grow 1 vial of HUVEC out to 15 population doublings?

  The amount of media needed per vial depends on the growth characteristics of the cells, the size of the TC vessels and the split ratios used, the frequency of media changes, the type of experiments you perform, etc. It is therefore difficult to give definite quantities. As a rough guideline, 1-2 bottles (500 ml each) are needed for 1 vial of HUVEC.

  Related Links and Documents

  • Endothelial Cell Growth Media
• Which cytokine concentrations does PromoCell recommend for activating M1/M2 macrophages?

  You may find all information regarding the activation and the cytokine concentrations in table 1 (page 5) of our Application Note.

  Related Links and Documents

  • Application Note
  • Macrophage Cell Culture
• What is the difference between PromoCell NHEK and NHEK GM3?

  The NHEK (primary human keratinocytes) are isolated in our serum-free Keratinocyte Growth Medium 2 (C-20011), the NHEK GM3 in our improved serum-free and BPE-free Keratinocyte Growth Medium 3 (C-20021). Both, NHEK and NHEK GM3 are available from single or from pooled donors isolated from the epidermis of juvenile foreskin or adult skin.

  Related Links and Documents

  • NHEK
  • NHEK GM3
• Can you drive organoid orientation (inward vs outward)?

  Yes. The orientation can be triggered by the use or lack thereof of ECM.

  Without the use of an ECM, the organoids will have a higher outward oriented ratio. You can find a protocol here https://pubmed.ncbi.nlm.nih.gov/30811997/, where cells were first embedded in ECM gel and afterwards ECM was dissociated, and free organoids were re-seeded in suspension without an ECM.

  * Please note that we have not tested this method in our labs and thus cannot guarantee it.

  Related Links and Documents

  • AppNote: Generation of Human Airway Organoids from Primary Cells
• What are raw materials or ancillary materials and how do these differ from pharmaceutical excipients?

  Raw materials come in contact with the cell or tissue product during manufacturing but are not intended to be part of the final product. For example, cell culture media and reagents can be used in research and production of cell-based drugs and therapies. In this case they are defined as raw materials. During drug, cosmetic, and cell therapy development, the quality of raw materials must be carefully considered as they may have an effect on the efficacy of the final product and subsequent safety for the patient [1].

  The nomenclature for raw materials differs between the regions. The terminology “raw material” is used by European regulators.  In other regions the synonymous term ancillary material is used.

  Raw materials or ancillary materials are commonly labeled as “not for use in clinical or diagnostic procedures” or “for ex vivo use only and not intended for human in vivo applications”.

  Pharmaceutical excipients are substances that are included in a pharmaceutical dosage form not for their direct therapeutic action, but to aid the manufacturing process, to protect, support or enhance stability, or for bioavailability or patient acceptability. They may further assist in the effectiveness and/or delivery of the drug in use and maintain the integrity of the drug product during storage.

  [1] Solomon J, Csontos L, Clarke D, Bonyhadi M, Zylberberg C, McNiece I, Kurtzberg J, Bell R, Deans R. Current perspectives on the use of ancillary materials for the manufacture of cellular therapies. Cytotherapy. 2016 Jan;18(1):1-12.

  Related Links and Documents

  • PromoExQ MSC Growth Medium XF
• In the past, we used MSC Growth Medium (C-28010) in our lab. Can I use MSC Growth Medium 2 (C-28009) instead? What is the difference?

  MSC Growth Medium 2 (C-28009) is an optimized medium formulation with reduced serum content to allow for more standardized culture conditions (considerably lower lot to lot variation).
  You can replace MSC Growth Medium by MSC Growth Medium 2. Coating of culture vessels is not necessary. We recommend to plate the cells (hMSCs from bone marrow, adipose tissue, or umbilical cord) at 4,000 cells/cm².

  Related Links and Documents

  • Human Mesenchymal Stem Cell Media
• Does PromoCell recommend a specific plasticware to use for the generation of dendritic cells from peripheral blood monocytes?

  Usually, we do not recommend specific plasticware but we have found that with PromoCell’s Dendritic Cell and Macrophage Generation Media, choice of plasticware can have a lot of an influence. For our Dendritic Cell Generation Media we recommend to use tissue culture vessels from BD Falcon^TM.

  Related Links and Documents

  • DC Generation Media
  • Application Note: Generation of moDCs
• What is the difference between DC Generation Medium XF (C-28052) and DC Base Medium XF (C-28054)?
  • DC Base Medium XF (C-28054) consists of 250 ml Basal Medium + SupplementMix. It does not include cytokines and must therefore be supplemented according to the user’s individual needs.

  • DC Generation Medium XF (C-28052) is ready-to-use and consists of 250 ml Basal Medium + SupplementMix + appropriate cytokines.

  Both, Base Medium XF and Generation Medium XF have a xeno-free formulation.

  Related Links and Documents

  • DC Media XF
• What’s the difference between PromoCell HUVECs and HUV-EC-C from ATCC?

  PromoCell HUVECs are freshly isolated from umbilical veins. They are cryopreserved at the end of primary culture. After revival, they can be propagated for at least 15 doublings and will senesce eventually.
  HUV-EC-C from ATCC is a hypodiploid human cell line of endothelial origin (umbilical vein). The modal chromosome number is 45 occurring in 72% of cells counted. The rate of polyploid cells is 15.8%. The cells have a life expectancy of 50 to 60 population doublings. This indicates that the cells are no longer “normal cells” but have undergone some degree of transformation.

  Related Links and Documents

  • HUVEC
• Will the hematopoietic progenitor cells proliferate in Hematopoietic Progenitor Cell Expansion Medium XF (C-28021) without Cytokine Mix E (C-39890)?

  For the expansion of hematopoietic progenitors (CD133+ cells, CD34+ cells), PromoCell has developed Hematopoietic Progenitor Cell Expansion Medium XF (C-28021), a serum-free and xeno-free formulation. The medium must be supplemented with either Cytokine Mix E (C-39890) or with the user’s own mixture of cytokines. Cytokine Mix E is a ready-to-use mix containing recombinant human TPO, SCF, flt3-ligand, and IL-3.
  The strong expansion of the progenitor cells in Expansion Medium XF + Cytokine Mix E persists for at least two weeks, resulting in a 200-300x increase of the cell number with only 20-30% differentiated cells.
  Note: If starting with CD133+ cells, the CD133 marker is getting lost during this expansion step. The resulting cells are D34⁺/CD38^–/CD133^–.

  Related Links and Documents

  • hCD34+ Progenitor Cells
  • Hematopoietic Progenitor Expansion Medium XF
• Can mycoplasma contamination be observed with the naked eye?

  No, mycoplasma can only be observed through electron microscopy. For highly sensitive detection of mycoplasma contamination, we recommend the use of PCR-based mycoplasma tests.