Have a question about one of our products? Check out our technical library for recently asked questions from other scientists around the world.
Technical Library
Recent Entries in Technical Library
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Where does PromoCell source the heart tissue for HCM isolation?
In most cases, the tissue comes from patients who underwent heart transplantation. We obtain a part of the explanted (not the transplanted) heart to prepare the cardiac myocytes.
In other cases, the tissue comes from LVAD (= Left Ventricular Assist Device) surgery.Related Links and Documents
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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
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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.
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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
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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- or vitronectin-coated TC vessels.
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Do all PromoCell HUF come from the myometrium or does the uterine location vary by lot?
All our Human Uterine Fibroblasts (HUF) are prepared from the myometrium.
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Which DC medium does PromoCell recommend for freshly isolated MNC or monocytes, which one for cryopreserved monocytes?
The PromoCell DC Generation Media have been developed for the easy and efficient generation of immature as well as fully mature myeloid dendritic cells from peripheral blood monocytes.
For freshly isolated mononuclear cells (MNC) and monocytes we recommend our DC Generation Medium XF (C-28052), for cryopreserved monocytes our DC Generation Medium (C-28050). When using DC Generation Medium (C-28050) with fresh MNC, the Monocyte Attachment Medium (C-28051) is needed in a first step for efficient adherence of the monocyte fraction.
Please see Instruction Manual, Application Note and the graph below for further details.Related Links and Documents
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What are the key points relating to proliferation, differentiation and culturing of HWP?
Our 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. The 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
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Why is the serum content of PromoCell’s specialized media so low?
FCS is a natural product consisting of many different components like salts, hormones, vitamins, trace elements, proteins, and enzymes. There can be large lot-to-lot variations between different serum batches regarding the concentrations of growth-promoting factors. The higher the serum content in a culture medium, the higher the impact of the variations on the cell culture system. For this reason, PromoCell has developed several serum-reduced and serum-free media where part of the serum has been replaced by more defined factors like cytokines, hormones or vitamins.Media with reduced serum concentrations have the benefit that they produce more standardized culture conditions over a long time span.
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Does PromoCell determine the percentage of HDLEC in its HDMEC lots?
No, we don’t determine the ratio of HDLEC and HDBEC in our HDMEC lots. From our experience, the percentage of HDLEC is highly lot-dependent and can vary between 5 and 60%.
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Can I use the Growth Medium instead of TNS to stop the trypsin when splitting the cells?
A concentration > 10% FBS is needed to completely inactivate the trypsin. As most of PromoCell’s growth media are serum-reduced or serum-free, the use of a trypsin inhibitor like TNS is highly recommended.
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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.
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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
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How can I calculate the population doubling time?
The population doubling time or generation time (tg) 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.
tg = t / nn: number of population doublings
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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
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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).
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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.
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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
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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.
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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.
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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
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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.
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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.
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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.
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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).
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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.
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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
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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.
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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.
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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.
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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.
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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
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How many cells do the cell pellets contain?
PromoCell cell pellets are made from > 1 million cells and are dissolved in 200 µl RNAlater.
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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.
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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.
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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.
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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
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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
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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.
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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.
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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.
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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
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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.
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At what passage do PromoCell supply the cell pellets?
The majority of PromoCell cell pellets (C-14**) are prepared 1 passage after thawing the cryopreserved cells.
Examples:
HUVEC and HUAEC correspond to P1 after thawing; therefore the pellets are frozen in P2.
SMC or keratinocytes or epithelial cells correspond to P2 after thawing; therefore the pellets are frozen in P3.
In contrast, our blood cells are cryopreserved directly after cell isolation. These pellets are prepared after thawing the cryovials with no further cultivation step. -
From what tissue do PromoCell isolate their HUtMEC (C-12295)?
Our HUtMEC are isolated from the middle layer of the uterine wall (myometrium). The tissue donors were not pre-treated with hormones.
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What is the composition of the Osteoblast Supplement (C-39615)?
Our Osteoblast Supplement consists of FCS (10 % [v/v] final concentration) which is specifically tested to support optimal growth of Normal Human Osteoblasts.
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Should the skeletal muscle cells be split after adding the Differentiation Medium (C-23061)?
After adding the Skeletal Muscle Cell Differentiation Medium, myoblasts will start to differentiate into myotubes and stop growing. The cells should not be split anymore. It is recommended rather to plate the SkMC into the needed vessels, (e.g. multiwell plates), prior to induction of differentiation and to perform studies directly on differentiated cells. If it is necessary for your tests to detach the myotubes and they are difficult to trypsinze, you can use a “rubber policeman”.
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Does PromoCell offer a special 3-D system to block de-differentiation or does PromoCell have special culture conditions that stabilize the chondrocyte phenotype?
PromoCell Chondrocytes (HCH) can be expanded in normal monolayer culture using our Chondrocyte Growth Medium (C-27101). The Medium consists of an optimized formulation and is supplemented with 10 % FCS. De-differentiation of chondrocytes is a known phenomenon observed during in vitro-culture after a period of approx. 2 weeks, but in vitro-culture is needed to expand the cells.
Once a suitable cell number is obtained, the monolayer system can be changed to a more complex 3-D system either by culturing the cells on substrates like alginate beads, gels, or degradable polymer scaffolds or by using 3-D spheroid culture. Using appropriate conditions, re-differentiation is triggered and the cells start producing cartilage-specific ECM again.
PromoCell does not supply a special culture system but we use 3-D spheroid culture and Alcian Blue staining to characterize our chondrocytes during quality control. The protocol is very similar to the one we use for chondrogenic differentiation of MSC.Related Links and Documents
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Does the PromoCell 3D Tumorsphere Medium XF (C-28070) maintain the stem cell character of cancer stem cells?
During our quality control we do not determine the presence or maintenance of respective stem cell markers in the tumorspheres. We use a functional approach instead, by culturing the tumorspheres over serial passages in C-28070. Tumorsphere formation requires the biological features of Anoikis resistance and self-renewal and therefore indicates the presence of cancer stem cells (CSC)/cancer initiating cells (CIC) in the spheres. 3D tumorsphere culture thus allows scientists to study the biology of CSCs without any background knowledge on CSC markers.
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Does PromoCell have any data regarding the cytokine profile and different markers of the M1/M2 macrophages after activation?
No, we do not provide data about the cytokine profile of our M1/M2 macrophages after activation and we do not provide any further data beyond the scope of our quality control.
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Can the Cytokine Mix E for the Hematopoietic Progenitor Expansion Medium XF (C-39891; 5 ml) be safely aliquoted?
Yes, it is possible to aliquot the 5 ml Cytokine Mix E (C-39891) into 5 x 1 ml.
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Can you control size / size distribution of organoids?
Possibly. The size of the organoids may depend on the access to nutrients in the gel. For reference, https://pubmed.ncbi.nlm.nih.gov/31173716/
For best results we recommend the following:
- Avoid nutrient gradients. We recommend using a small, drop-like BME gel bead and high volumes of medium. Change the medium every day as we’ve observed that less frequent medium changes results in yellow culture medium indicative of a pH shift likely from an increase in cellular metabolism.
- For optimal cell distribution, it is important to control the “gelling” or setting of the ECM gel. Therefore, the temperature is critical! BME is liquid in the cold (4°C) and solidifies at 37°C. The gelling must be done in a very short time, otherwise the cells in the BME would sink to the bottom.
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What is the EXCiPACT™ GMP certification?
Pharmaceutical excipient production should be carried out in accordance with the principles defined in the Good Manufacturing Practices (GMP). With that in mind, a group of industry experts including EFCG, IPEC and PQG worked together to develop the EXCiPACT™ certification scheme for pharmaceutical excipient suppliers.
EXCiPACT™ is a non-profit organization that oversees and operates an independent, high-quality, third-party certification system. This certification system is available to pharmaceutical manufacturers and distributors around the world.
To achieve EXCiPACT™ GMP certification, we demonstrated that our pharmaceutical excipients are manufactured according to the GMP guidelines, as well as ensuring compliance in our raw material supply chain, production environment, production processes and storage conditions.
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I would like to know if the Lymphocyte Separation Medium (C-44010) is endotoxin tested.
Yes, our Lymphocyte Separation Medium 1077 is endotoxin-tested. The specification for the product is < 10EU/ml.
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Is the PromoCell Keratinocyte Growth Medium 2 suitable for growth of primary mouse keratinocytes?
Yes, primary mouse keratinocytes grow well in this media when you reduce the CaCl2 concentration to 0.025 mM – 0.05 mM CaCl2 (optimal Ca concentration for primary human keratinocytes is 0.06 mM).
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When do I need PromoCell’s Monocyte Attachment Medium (C-28051)?
The Monocyte Attachment Medium allows for the efficient adherence selection (within 1 hr) of monocytes from freshly isolated mononuclear cells while maintaining optimal cell health. The Time-consuming and costly immunomagnetic purification of monocytes prior to DC generation is not necessary and can be skipped when using this medium.
.Related Links and Documents
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How long does it take to detach primary cells with accutase (C-41310)?
Most primary cells detach within 5-10 min at 37°C. Inactivation isn’t required but we recommend to centrifuge the cell suspension to remove accutase and EDTA before replating the cells.
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Is it possible to transfect PromoCell Normal Human Cells?
Yes, it is possible to transfect our normal human cells. In general, primary and normal cells are much harder to transfect than immortalized cell lines. Apart from the cell type, successful transfection also depends on the culture’s age and density at transfection, the vector used, the purity of the nucleic acids, the composition of the transfection medium, and the experimental conditions.
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What’s the stability of HPC Expansion Medium XF (C-28021) after addition of Cytokine Mix E (C-39890)?
The medium supplemented with Cytokine Mix E is stable for 2 weeks if stored protected from light at 2-8°C.
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Is it necessary to heat inactivate FCS for use with Normal Human Cells?
The practice of heat inactivation was originally developed when only serum from adult animals was available. Adult serum contains high serum complement which may destroy cells under certain conditions. Heating serum (30 min, 56°C) is intended to inactivate the complement. Today, serum is often heat-inactivated without any evidence of beneficial effect. When using FCS (fetal calf serum), heat inactivation is not necessary for most cell lines or cell types. PromoCell does not use heat-inactivated FCS for the preparation of their growth media.
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To what extent will the CD34+ Progenitor Cells proliferate in Hematopoietic Progenitor Cell Expansion Medium XF (C-28021)?
For optimal cell growth, the human CD34+ Progenitors (C-12921) are cultured in PromoCell Hematopoietic Progenitor Cell Expansion Medium XF (C-28021) supplemented with Cytokine Mix E (C-39890) or with any other cytokine cocktail suitable to achieve optimal cell expansion. Cytokine Mix E contains recombinant human TPO, SCF, flt3-ligand and IL-3. The strong expansion of CD34+ cells will persist for approx. 2 weeks. The expansion factor is usually between 75 and 200-fold.
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What are the criteria for selection or exclusion of peripheral blood donors? Is the peripheral blood that PromoCell use to isolate blood cells (MNC-PB, monocytes) derived from completely healthy donors?
The following criteria are applied for blood donations:
a) Donors with light infection like a cold or cough are excluded for one week
b) Donors with infections with a temperature above 37.9°C and/ or antibiotics therapy are excluded for 4 weeks
c) Blood pressure: exclusion only if systolic blood pressure is < 100 mm Hg or > 180 mm Hg or if diastolic blood pressure is > 100 mm Hg. No exclusion if blood pressure is drug treated and in acceptable range.
d) High cholesterol: no exclusion even though medication is used
e) Diabetes: exclusion if diabetes type I or use of insulin
f) Steroid use: exclusion for 4 weeks after application
g) Cancer: exclusion
h) Other chronic diseases: exclusion depends on the type of disease (for example chronic heart disease, autoimmune disease)Related Links and Documents
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What medium should I use to grow or differentiate PromoCell Human Stem and Blood Cells?
The recommended media for a particular cell type are specified on the respective product page (“Recommended Products”) and can be found in the Manual belonging to the cells.
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From what tissue do PromoCell isolate their HPMEC (C-12281)?
Our HPMEC are isolated from peripheral lung tissue of adult donors.
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How should I thaw the PromoCell normal human cells to obtain a high viability?
General protocol for recovery of anchorage-dependent primary cells:
Remove vial from liquid nitrogen, transport it on dry ice to the cell culture lab. Thaw in a 37°C waterbath for approx. 2 min, until it is just defrosted. Keep the vial immersed in water until just below the screw cap during thawing and only remove it shortly after approx. 90 sec to check the progress.
Carefully disinfect the vial with plenty of 70% EtOH under the laminar flow hood and aseptically transfer the thawed suspension into an appropriate TC dish with growth medium (pre-warmed in the incubator for > 30 min).
The cells usually attach within a few hours. Perform media change after 24 hrs at the latest to remove residual DMSO from the freezing media. Additional information can be found in the instruction manuals of our cells.Related Links and Documents
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Are the PromoCell Skeletal Muscle Cells (C-12530) in a differentiated or undifferentiated state?
Our SkMC are proliferating myoblasts that have retained the capacity to differentiate. Upon withdrawal of serum and growth factors, differentiation is induced and the cells form multinucleated syncytia.
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After how many passages in monolayer culture do the chondrocytes start to de-differentiate?
Mature chondrocytes from articular cartilage are postmitotic producers of extracellular matrix components like collagen types II, IX, and XI and proteoglycans. Upon release from the tissue of origin and seeding in monolayer culture, cells re-enter the cell cycle and proliferate. After a period of approx. 1-3 weeks, they will gradually start dedifferentiation and will adopt a fibroblastic phenotype. Dedifferentiation can be detected by decreased collagen type II or increased collagen type I expression, as well as by the appearence of the fibroblast marker Thy-1/CD90. Re-differentiation can be induced by changing from conventional monolayer culture to a 3-D culture system.
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Are there PromoCell specialized media / primary human cells alternatives to other supplier’s products?
Please see our attached alternative product guides for media and cells.
Please note: Our media do not contain antibiotics. For optimal cell growth, we recommend to refrain from using antibiotics. However, when a sterile environment cannot be 100% ensured, it may be advisable to add antibiotics to your media so as to protect the cultures from potential microbial infections.
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Do you recommend a certain negative control for the activated macrophages to compare the cytokine release with?
Even non-activated macrophages do release a certain amount of cytokines. Furthermore, you would have to be sure that the release of a certain cytokine is a direct consequence of the activation. Therefore, we do not think it is possible to have a general negative control for the cytokine release.
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How long do I need to activate my macrophages? Can I stop adding activation factors after 24 hours?
The activation of macrophages as such is complete after 24 hrs. However, to maintain the activation status over a longer period of time (i.e., several days), fresh activation factors should be added with every medium change.
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Why use ROCKi for organoid formation?
ROCKi is known to enhance the proliferation of epithelial cells or keratinocyte progenitors and can improve cell survival via conditional reprogramming and the effect is reversible. It is also known to enhance the seeding efficiency on plastic.
This ROCKi conditional reprogramming has also been observed in airway EpCs and cultured organoids:
For our human airway organoids, the use of Y-27632 ROCKi is optional as organoids can still form without it. However, the 3D system has some potential pitfalls like choosing the right plastic or the right ECM. The use of ROCKi may help to keep the system more robust.
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Can I access your ISO and EXCiPACT™ Certification?
All our products meet the strictest European and international ethical standards, and our Quality Management System is certified according to ISO 9001:2015 certification and the EXCiPACT™ GMP standard. These certifications ensure that we consistently provide products and services that meet researchers’ and applicable statutory as well as regulatory requirements while also covering the GMP requirements according to NSF/IPEC/ANSI 363.
The following documentations and certification can be directly downloaded on our website
- Quality Policy Statement
- ISO 9001:2015 certificate
- EXCiPACT™ certificate
- ANSI 363-2016 certificate
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Can I use the 3D Tumorsphere Medium XF for mouse cell lines?
Yes, we have received a customer feedback that our 3D Tumorsphere Medium XF (C-28070) has been successfully used for tumorsphere formation of mouse cell lines.
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Looking at the detachment protocol of PromoCell’s Macrophage Detachment Solution, I observed that we need to add HSA to the PBS. Can I use FBS instead? Also, why are we adding 2 mM EDTA to the PBS?
- FBS (or BSA) can be substituted for HSA, however we do not recommend this as the FBS will lead to unpreferable immunologic stimulation of human macrophages.
- The addition of EDTA to the PBS is to further augment the “anti-clumping” activity of the Ca2+/Mg2+ free PBS.
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What is the difference between DC Generation Medium Ready-to-use (C-28050) and DC Base Medium (C-28053)?
- DC Base Medium (C-28053) consists of 250 ml Basal Medium + SupplementMix. It does not include cytokines and must therefore be supplemented according to the user’s individual needs.
- DC Generation Medium (C-28050) is ready-to-use and consists of 250 ml Basal Medium + SupplementMix + cytokines.
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Do the PromoCell media contain L-glutamine?
Yes, the specialized PromoCell media already contain the optimal amount of L-glutamine. Please don’t add extra L-glutamine as this can be toxic for the cells.
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What’s the difference between trypsin and accutase?
Both, trypsin and accutase represent mixtures of different proteolytic enzymes. Trypsin is prepared from porcine pancreas, accutase from invertebrates. Accutase can replace trypsin for the detachment and dissociation of anchorage-dependent cells from surfaces and can also be used on suspension cells to reduce clumping in preparation for counting. The advantages of accutase over the traditional trypsin treatment are that it is more gentle and less damaging to cells (leading to increased viability) and does not contain any mammalian or bacterially derived proteins. Accutase is more thermolabile than trypsin and usually doesn’t require an inactivation step.
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Why are most of the soluble receptors not produced in E. coli?
Soluble receptors are post-translationally modified and generally contain disulfide bonds and glycosylation sites. If produced in E. coli the proteins don’t show any biological activity.
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What is the DMSO concentration in Cryo-SFM?
The DMSO concentration in our Cryo-SFM is 10%.
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I am interested in purchasing PromoCell Human Mononuclear Cells (C-12907). Could you please let me know whether you have tested them for platelet contamination? If yes, which markers / procedure have you tried?
During the isolation of our Human Mononuclear Cells (hMNC), we first of all pay attention to thoroughly discard the platelet-containing fraction before we aspirate the MNC containing-interphase of the Ficoll gradient. The harvested MNC fraction is then subjected to several washing steps to remove potential remaining platelets. Finally, the cell preparations are verified by microscopy to be largely free of platelet contamination.
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Can I starve PromoCell HUVECs?
We have many customers who perform starvation with our HUVECs. Most of them use Endothelial Cell Basal Medium supplemented with FCS (0.5-1% for shorter periods; 5-20% for 24-48 hrs). The cells have to be in a good condition and the experiment should be terminated soon after starvation. Prolonged periods will induce apoptosis.
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What media should I use to grow PromoCell Normal Human Cells?
The recommended Growth Media are specified in the Manual belonging to the cells and can be also found on the product page of the cells (“Recommender products”).
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What are the precise localizations of both, juvenile and adult HDMEC?
Juvenile HDMEC (C-12210) are isolated from the dermis of foreskin. Adult HDMEC (C-12212) are isolated from different regions. The localizations include cheek, temple, breast, upper arm, and labia. Please contact our Technical Customer Support if you need HDMEC from a particular part of the body.
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My Normal Human Cells have changed their morphology. What could be the reason?
Your observation can be either ascribed to a change of the culture medium, to a cross-contamination with another cell type, or to differentiation or senescence. The attached trouble shooting guide should help you to identify the reason for your observation.
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Can I mix the SkMC Supplement with the Skeletal Muscle Cell Basal Medium and freeze some down for later use? If so, how long can it remain frozen?
The PromoCell Basal Media must be stored between 4-8°C and should not be frozen, as this can lead to precipitations. The same is true after addition of the supplements: the complete medium has to be kept at 4-8°C.
If you prefer to make up smaller volumes of complete medium, you can aliquot the Supplement Mix and refreeze those aliquots at -20°C until use. This way you can extend the period in which you can use the supplemented media.Related Links and Documents
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When we buy chondrocytes from PromoCell, is it possible to make sure they are articular chondrocytes, i.e. from knee joint?
All our chondrocytes (C-12710) are isolated either from knee joint or femoral head. If you need chondrocytes specifically from either localization, please contact our Technical Customer Service before placing your order. They will send you the available lot numbers from the localization that you are looking for.
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Which medium does PromoCell recommend for endothelial cell transfection ?
There are several factors that can influence successful transfections, e.g. viability and density of the cells, choice of the transfection reagent, quality and type of the transfected molecules (plasmids, siRNA, oligonucleotides), as well as the culture medium and supplements used.
When using PromoCell Endothelial Cell Growth Media for cell transfection, please follow the instructions below:Heparin, which is included in our Endothelial Cell Growth Medium (C-22010/C-22110), Medium 2 (C-22011/C-22111) and Medium MV (C-22020/C-22120) may reduce the transfection efficiency. We therefore recommend to use our heparin-free Endothelial Cell Growth Medium MV2 (C-22022/C-22121). Alternatively, you may use Endothelial Cell Growth Medium Kit (C-22110), Medium 2 Kit (C-22111), or Medium MV Kit (C-22120) without adding the ECGS/heparin supplement to the Basal Medium.
Please note: Before and after transfection, the cells should be cultured in complete Growth Medium including heparin to ensure optimal growth.
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Following the recommended seeding density for your M1 macrophages (100,000 cells per cm2), I would need too many cells to have a complete 96- or 384-well plate. This is too expensive for me, can I reduce the number of cells per cm2?
The recommended seeding density with 100,000 cells per cm2 is needed for a confluent cell layer as the cells do not proliferate.
However, you can reduce the seeding density by the factor 3 to 5 and the macrophages are still viable.Related Links and Documents
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Why are PromoCell products “for in vitro research use only”?
The basis for the intended use of our products is defined in our Terms & Conditions under the chapter “Use of Goods”.
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How do you set up high throughput systems with organoids? Only in 96 well?
It is possible to culture the airway organoids in 96-well U-Bottom plates for suspension cells (e.g., Greiner Bio-One # 650185).
A detailed protocol for the use of 96-well plates can be found in our AppNote. We have not tested 384-well plates or other commercially available plates.Related Links and Documents
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Where can I find the supporting documents and the material safety data sheet for your products?
Using the product lot number the Supporting Documents can be downloaded at: www.promocell.com/eq-certificates
The Material Safety Data Sheet (MSDS) can be directly downloaded from our website on the product page”
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