Technical Library

Recent Entries in Technical Library

• Can you please provide me a protocol for the expansion of CD34+ Progenitor Cells in PromoCell Expansion Medium XF?

  Short protocol:

  • Thaw the cells (C-12921) for 2 min in a 37°C waterbath. Dilute in 9 ml of complete HPC Expansion Medium XF (+ Cytokine Mix E) and count the cells
  • Spin down for 10 min at 240xg, aspirate the supernatant, resuspend the pellet at 20,000 cells/ml HPC Expansion Medium XF
  • Plate in an appropriate suspension culture vessel and incubate the culture for 2-3 days at 37°C and 5% CO₂
  • Then double the media volume by adding fresh complete medium, e.g., 4 ml  suspension culture + 4 ml fresh medium (= 8 ml)
  • Incubate the cells for an additional 10-12 days by performing a partial medium change every 2-3 days
    Example partial medium change: For a culture volume of 8 ml, spin down the cells, aspirate and discard 4 ml of the supernatant, resuspend the cells and add 12 ml of fresh complete medium (= 16 ml).

  Depending on the donor a total expansion factor of approximately 300-1,000 fold (CD34⁺ plus CD34^– cells) and an expansion of the CD34⁺ population by approx. 50–200 fold can be expected when using complete HPC Expansion Medium XF with Cytokine Mix E.

   

  Related Links and Documents

  • App Note: Expansion of primitive Hematopoietic Progenitor Cells
  • Hematopoietic Stem Cell Culture
• We use Macrophage Generation Media from Promocell to get differentiated macrophages from fresh PBMC. Would it be okay if the attachment step with the Monocyte Attachment Medium is done overnight instead of 1.5 h?

  We strongly advise against overnight incubation.

  The Monocyte Attachment Medium does not contain cytokines/survival factors for the monocytes. If the cells remain in the Monocyte Attachment Medium for longer than 2 hours, they will go into apoptosis and die. The lymphocytes will survive longer. Some of them will attach after such a long time and then cannot be washed off.

  Therefore, overnight attachment is absolutely inappropriate. However, if time is short, monocyte attachment can be shortened to 1 hour. To do this, it is best to add Monocyte Attachment Medium to the culture vessels in advance and equilibrate it in the incubator. Remove the vessels from the incubator only briefly to add the appropriate amount of concentrated PBMC suspension. Then, even after 1 hour, most of the monocytes are attached.

  Related Links and Documents

  • Monocyte Attachment Medium
• Can I use a serum-free medium as negative control in the chondrogenic MSC differentiation? I would like to replace the recommended DMEM+10% FCS.

  You could probably use our MSC Chondrogenic Differentiation Medium without inducers (C-28014). It is also serum-free and the same as C-28012, just without chondrogenic inducers.

  Related Links and Documents

  • App Note: Chondrogenic Differentiation and Analysis of MSC
  • Mesenchymal Stem Cell Chondrogenic Differentiation Media
• Is it possible to thaw and re-freeze the Cytokine Mix M1 (C-39894) and M2 (C-39895)?

  The Cytokine Mix M1 and M2 should not be subjected to further freeze/thaw cycles.

  Related Links and Documents

  • M1-Macrophage Generation Medium XF
  • M2-Macrophage Generation Medium XF
• Can I use a bead bath to thaw PromoCell normal human cells?

  Based on negative feedback we have received from customers using bead baths, we strongly discourage the use of bead baths to thaw our cells. It can lead to reduced viability or significantly slower growing cells.

  If you don’t have a “normal” water bath but only a bead bath in your lab, thaw the vial in a beaker of water in the bead bath. Ensure the water is heated to exactly 37°C using a thermometer placed in the warmed water. Be sure to hold the vial in your hand, and not in a floater, as described in the thawing protocol.

• Do you find that the domes spread a lot on the Nunclon Sphera plates? Do they remain attached for the entire 3-4 week culture period?

  To avoid the spreading of the matrix and to have nice drop-like domes, it is crucial to preheat the plate for 2h at 37°C and to work fast when transferring the gel to the wells. Placing a warming plate under your laminar air flow hood when transferring the gel-cell-mix to the preheated plate may help.

  From our testing, we found that Nunclon Sphera wells were not ideal for our organoid system as the dome did not stay adhered, but it was able to move on the bottom and the dome became misshaped overtime. However, the altered shape of the dome did not affect the organoids and were successfully cultured for 4 weeks. If movement is not an issue for the user, we recommend that extra caution be taken during media changes by holding the plate at an angle and carefully pipette to remove old medium. Aspirating with vacuum suction can damage or destroy the dome.

  Related Links and Documents

  • AppNote: Generation of Human Airway Organoids from Primary Cells
• 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

  • AppNote: Generation of Human Airway Organoids from Primary Cells
• 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:

  • https://pubmed.ncbi.nlm.nih.gov/31871466/
  • https://pubmed.ncbi.nlm.nih.gov/35169685/

  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.

  Related Links and Documents

  • AppNote: Generation of Human Airway Organoids from Primary Cells
• 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.

  Related Links and Documents

  • AppNote: Generation of Human Airway Organoids from Primary Cells
• 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
• 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
• 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
• 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
• 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
• Why is PBS added in the last step of the Alizarin Red staining protocol (“Osteogenic differentiation and analysis of MSC”)? Should the PBS be aspirated before analysis?

  The PBS buffering enhances the Alizarin Red staining (precipitation of the dye) and makes it more intense.
  Leave the PBS on the cells after staining/washing and analyze the sample immediately, as the dye may bleed upon prolonged storage without embedding.

   

  Related Links and Documents

  • Application Note: Osteogenic Differentiation and Analysis of MSC
• What are the disadvantages of the prophylactic use of antibiotics in cell culture?

  The use of antibiotics creates a false sense of security and allows users to develop poor aseptic techniques. This leads to low-level contamination with partially resistant bacteria occurring but being overlooked for a time. This then leads to cells with undetected contamination being cultured for extended periods of time, increasing the risk that contamination will spread throughout the laboratory and eventually antibiotic-resistant strains of bacteria may develop.
  Mycoplasma infections can also occur more easily, as they are often introduced along with contaminants such as bacteria and fungi.
  Last but not least, antibiotics are known to have negative effects on the metabolism of eukaryotic cells – more details on this topic can be found in our blog article “Antibiotics in cell culture: friend or enemy”.

  Related Links and Documents

  • Blog article: Antibiotics in Cell Culture: Friend or Enemy?
• I have accidently stored the Cryo-SFM at -20°C. Can the product still be used in this case?

  According to the product manual, Cryo-SFM should be stored at 4-8°C. However, since this solution is used to freeze cells in liquid nitrogen, we assume that storing Cryo-SFM once at -20°C should not have a negative impact on the product quality. After thawing, please store it at 4-8°C as recommended.

  Related Links and Documents

  • Freezing Medium Cryo-SFM
• How can I avoid precipitates when preparing my Mesenchymal Stem Cell Adipogenic Differentiation Medium 2?

  The supplement should be at room temperature when added to the MSC Adipogenic Basal Medium 2. It may also be beneficial to invert the tube a few times to bring precipitates back into solution.

  Please note: It is not recommended to filter the basal medium, supplements, or complete medium, as components that induce or promote differentiation may be removed, resulting in a low differentiation rate when using the medium.

  Related Links and Documents

  • Mesenchymal Stem Cell Adipogenic Differentiation Medium 2
• Are there any differences in the cultivation protocol when cultivating MSCs compared to other cell types?

  Yes, there are a few differences:
  – We recommend replacing the MSC Growth Medium XF (C-28019) 3-4 h after seeding, as opposed to 16-24 hours after seeding for most other cell types/growth media.
  – When MSC Growth Medium XF, MSC Neurogenic Differentiation Medium, MSC Adipogenic Differentiation Medium 2 or MSC Osteogenic Differentiation Medium are used, flasks have to be coated with 10 µg/cm² (human or bovine) fibronectin according to the instruction manual.
  – We strongly recommend using Accutase (C-41310) for cell detachment instead of Trypsin. If Trypsin is used, contact time should not exceed 2 min.

   

  Related Links and Documents

  • Mesenchymal Stem Cell Culture
• After thawing the supplements, I see some precipitation. Is this normal?

  Light flocculation may be seen upon thawing the supplements containing ECGS/heparin or BPE. This does not affect the activity of our media.
  Optionally, the precipitate can be removed by centrifugation under sterile conditions.

  We recommend to thaw the supplements (SupplementMix or SupplementPack) at 15-25°C.

• We are working with PromoCell MSCs and MSC Adipogenic Differentiation Medium. Do you have any information on a comparison between Sudan III and Oil Red O and if Sudan III is more efficient at labeling lipid vesicles?

  You can also use Oil Red O to stain lipid droplets.  At PromoCell, we used to use Oil Red O as well, but switched to Sudan III some time ago for organizational reasons.

   

  Related Links and Documents

  • Application Note: Adipogenic Differentiation and Analysis of MSC
  • Human Mesenchymal Stem Cells
  • Mesenchymal Stem Cell Adipogenic Differentiation Medium 2
• Are PromoCell HPAEC and HPASMC obtained from the proximal or distal part of the pulmonary artery?

  Our HPAEC and HPASMC are isolated from the proximal pulmonary artery.

  Related Links and Documents

  • Human Pulmonary Artery Endothelial Cells
  • Human Pulmonary Artery Smooth Muscle Cells
• 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”.

   

• 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.

  Related Links and Documents

  • Application Note: Assay-ready adherent cultures of fully functional human primary macrophages
  • Macrophage Cell Culture
• 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.

  Related Links and Documents

  • Cytokine Mix E
• 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
• 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
• 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
• 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
• 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
• I am performing MSC chondrogenic, adipogenic, and osteogenic differentiation and am staining the cells. The protocol says to use Saccomanno Fixation Solution. Is there an alternative method to fix the cells because I don’t have Saccomanno Fixation Solution?

  Yes, you can use 4.5% neutral buffered formalin. Paraformaldehyde should work as well.

   

  Related Links and Documents

  • Mesenchymal Stem Cell Culture
• Is PromoCell’s Cryo-SFM produced under GMP standard?

  No, our Freezing Medium Cryo-SFM (C-29910) is not produced under GMP standard. It is for in vitro research use only and not appoved for diagnostic or therapeutic procedures.

  Related Links and Documents

  • Freezing Medium Cryo-SFM
• Is it possible to differentiate M1 macrophages from PBMC in 96-well plates?

  At PromoCell, we have not tested macrophage differentiation from PBMC in 96-well plates, but we know from users that it is possible.

  According to a customer the mononuclear cells differentiate very well in the 96-well format. A plating density of 1 million PBMCs (without prior determination of monocyte content) per well has been shown to be optimal. The working volume in a 96-well plate is usually 100 µl.

  Related Links and Documents

  • M1-Macrophage Generation Medium DXF
• Can the medium for M2 macrophages be switched into RPMI or RPMI + M-CSF after differentiation?

  Unfortunately, we did not test this in our hands, and it must be tested by the customer. In fact, our medium is completely different from RPMI and therefore we cannot predict if this is working. We only know the successful long-term culture from our system with our media.

  Related Links and Documents

  • M2-Macrophage Generation Medium DXF
• Why does the macrophage differentiation in the PromoCell protocol take 10 days? In the literature or following the feedback from other researchers, it usually only takes 7 days.

  M1 / M2 polarization also takes seven days in the PromoCell system but the protocol contains two more days for optional macrophage activation. If you only want non-activated M1 / M2 macrophages, the process is usually completed after 7 days.
  Nevertheless, PromoCell does not recommend shortening the 10-day protocol because you actually get a plus in viability and cell yield (due to the re-attachment of floating cells) on day 8-10 due to the media change.

  Related Links and Documents

  • Application Note: Differentiation of M1- or M2-Macrophages
  • M1-Macrophage Generation Medium DXF
  • M2-Macrophage Generation Medium DXF
• I differentiated M1 macrophages from self-sourced PBMCs following your differentiating protocol. In the final cell culture, I can see small cells attached to the macrophages which are CD3+ (T-cells). What is the reason for the T-cell contamination after the differentiation?

  The reason for the higher number of lymphocytes in the macrophage culture is probably due to an insufficient washing step during the purification of the monocyte via adherence. The 3 washing steps in our protocol are essential to receive a monocyte population of over 90%.

  Related Links and Documents

  • Application Note
  • M1-Macrophage Generation Medium DXF
• How long do I have to activate the cryopreserved macrophages to measure cytokine release?

  In general, we recommend activating the cells for 24 hours or at least over night for all kind of activations. If the activation is not optimal in your experimental setting, you can increase or decrease the activation time accordingly.

   

  Related Links and Documents

  • Application Note
  • Macrophage Cell Culture
• I would like to analyze the macrophages by microscope and I would therefore have to plate the cells on fibronectin-coated glass. Did you test if the macrophages grow on fibronectin-coated glass?

  We did not test if the macrophages attach on fibronectin-coated glass.

  Related Links and Documents

  • Macrophage Cell Culture
• 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

  • Macrophage Cell Culture
• 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.

  Related Links and Documents

  • Macrophage Cell Culture
• 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.

  Related Links and Documents

  • Macrophage Cell Culture
• 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
• 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
• 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
• 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
• 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
• The components of my DetachKit show different colors. Is this normal or does it affect their quality?

  The components of the PromoCell DetachKit may arrive on occasion with a non-uniform color appearance. This phenomenon is known by PromoCell’s Quality Assurance. It is reversible and does not influence the quality of the product.

  Related Links and Documents

  • PromoCell DetachKit
  • PromoCell DetachKit 2
• Can I defrost Accutase Solution, prepare aliquots and refreeze them?

  Yes, Accutase Solution can be defrosted, aliquoted, and then refrozen.

  Defrosting: Accutase should be defrosted overnight in the refrigerator or placed in a tub of cold tap water.  Do not defrost in a 37°C water bath.
  Stability: Once thawed, it is stable for at least 2 months in the refrigerator if stored promptly after use.

  Related Links and Documents

  • Accutase Solution
• Is it possible to refreeze the hCD34 progenitor cells after having amplifed them in PromoCell Hemaotopoietic Progenitor Cell Expansion Medium XF?

  Yes, it is possible to refreeze them.

  Related Links and Documents

  • CD34+ Progenitor Cells
  • HPC Expansion Medium XF
• Why are the cryopreserved macrophages abbreviated as hMDM-GMCSF(-) and hMDM-MCSF(-), respectively? What does the (-) stand for?
  • hMDM-GMCSF is the abbreviation for human monocyte-derived macrophages. They are polarized [⇒ differentiated with GM-CSF] but non-activated [⇒ (-)] M1 macrophages. 
  • hMDM-MCSF is the abbreviation for human monocyte-derived polarized [⇒ differentiated with M-CSF] but non-activated [⇒ (-)] M2 macrophages. 

  The macrophages can be seeded into all kinds of TC vessels. After plating, they can be maintained as biologically functional adherent cultures for several weeks.Optionally, user-customizable activation of the cells can be performed. For details, please see attached Application Note, page 2, Fig. 3 and page 5, Tab. 1).

  Related Links and Documents

  • Application Note: Assay-ready adherent cultures of fully functional human primary macrophages using cryopreserved cells
  • Cryopreserved Macrophages
  • Macrophage Generation Media DXF
• If I isolate fresh CD14-monocytes for M1/M2 macrophage generation, can I culture them for a period of time before differentiation?

  This is not advised. Please seed the freshly isolated CD14-monocytes immediately in the Monocyte Attachment Medium. Adding a culturing step will change the biological characteristics of monocytes very rapidly.

  Related Links and Documents

  • Application Note: Differentiation of M1- or M2-Macrophages from PBMC/Monocytes
  • Monocyte Attachment Medium
• Can you provide a list of cell lines that have been successfully used for tumorsphere formation with the PromoCell 3D Tumorsphere Medium XF?

  The following cell lines have been tested at PromoCell to form tumorspheres in 3D Tumorsphere Medium XF (C-28070):

  • U-87 MG
  • MCF-7
  • MDA-MB-231
  • HT-29
  • HT1080
  • HepG2
  • A-549
  • Panc-1
  • LNCaP
  • A-431

  ⇒ For more details, please view the attached Application Note.
  In addition, we have received customer feedbacks for the following cell lines:

  • HCT-116 (human colorectal carcinoma cell line)
  • Capan-1 (human pancreatic adenocarcinoma cell line)
  • PC3 (human prostate cancer cell line)
  • C42B (osteotropic prostate cancer cell line)
  • NCI-H23 (human lung epithelial adenocarcinoma cells)
  • IMR-32 (human neuroblast cell line)
  • A818-6 (human pancreatic ductal adenocarcinoma cell line)
  • HEK293 (human embryonic kidney cells)
  • Calu-1 (non-small-cell lung cancer cell line)

  Related Links and Documents

  • Application Note: Tumorsphere Culture of Cancer Stem Cells (CSC) with the PromoCell 3D Tumorsphere Medium XF
  • 3D Tumorsphere Medium XF
• Can PromoCell MSC Growth Medium 2 (C-28009) be used for Mesenchymal Stem Cells from rat?

  Yes, we have received a customer feedback that our MSC Growth Medium 2 also works for rat MSCs.
  The rat cells grow nicely in this medium and have a good viability.

  Related Links and Documents

  • MSC Growth Medium 2
• 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.

  Related Links and Documents

  • Manual: Endothelial Cell Growth Medium/Medium 2
  • Manual: Endothelial Cell Growth Medium MV/ MV2
  • Cell Transfection reagents
• 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.

  Related Links and Documents

  • Alternative Product Guide - Primary Human Cells: Lonza® – PromoCell
  • Alternative Product Guide - Primary Human Cell Media: Lonza® – PromoCell
  • PromoCell's packaging systems: Medium (Ready-to-use) vs. Medium Kit
• 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
• 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.

  Related Links and Documents

  • Product Manual: 3D Tumorsphere Medium XF
  • Application Note: Tumorsphere Culture of Cancer Stem Cells (CSC) with the PromoCell 3D Tumorsphere Medium XF
  • Application Note: Determination of the Tumorsphere Formation Efficiency (TFE) with the PromoCell 3D Tumorsphere Medium XF
• 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
• 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
• When should I use phenol red-free basal media for cell culture?

  It has been shown that phenol red has estrogenic properties. Phenol red-free media are therefore generally used in studies evaluating steroid hormone action in cultured, estrogen-responsive cells (Berthois et al., 1986). Furthermore, phenol red can also interfere with some analytical methods like photometric analyses.

  Related Links and Documents

  • Berthois et al.; Proc Natl Acad Sci U S A. 1986 Apr;83(8):2496-500
• Where can I find the composition of your basal media?

  The formulation of our basal media is proprietary information. If you need to know the concentration of a particular component for your experiments, please contact the PromoCell Technical Customer Service.

• Where can I find the composition of the supplements?

  The qualitative and quantitative composition of the supplements can be found on our website and in the data sheets of the specialized media. When there is no such information specified, the composition of the supplements is confidential.

• What antibiotics concentration should I add to the PromoCell media?

  If antibiotics are deemed necessary the recommended final concentrations are:
  100 U/ml penicillin + 100 µg/ml streptomycin or
  50 µg/ml gentamicin + 50 ng/ml amphotericin B

  Please note: Addition of antibiotics can reduce the growth rate of the cells.

• Are the Renal Epithelial Cells isolated from proximal or distal tubuli?

  PromoCell provides two types of Renal Epithelial Cells: Human Renal Epithelial Cells (HREpC) and Human Renal Cortical Epithelial Cells (HRCEpC).
  HREpC are isolated from the adult kidney and stain positive for cytokeratin. They comprise a heterogeneous population of renal epithelial cells. HRCEpC are isolated from the cortex of the kidney and comprise cells from proximal and distal tubuli. They also stain positive for cytokeratin.

  Related Links and Documents

  • Epithelial Cells
• How does PromoCell characterize their kidney cells (HREpC; HRCEpC) ?

  Our kidney cells are characterized by their epithelial cell morphology and by cytokeratin expression using a pan-cytokeratin antibody.

  Related Links and Documents

  • Renal epithelial cells
• What is the difference between PromoCell’s HREpC and HRCEpC?

  Our HREpC (C-12665) comprise a heterogenous mixture of renal epithelial cells isolated from cortex and medulla. The HRCEpC (C-12660) are derived from renal cortex only.

  Related Links and Documents

  • Renal Epithelial Cells
  • Schematic structure of a kidney
• What 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
• What is the differentiation ratio that can be achieved with PromoCell’s subcutaneous HWP?

  Most of our subcutaneous preadipocyte lots achieve > 80-90% differentiation when differentiation is induced at P2 (directly after thawing).
  We generally recommend using cells for differentiation tests that haven’t undergone more than 4-5 doublings (a maximum of 1 passage after thawing), as the differentiation ratio will decline with the age of the cells.

  Related Links and Documents

  • Human White Preadipocytes
  • Preadipocyte Growth Medium
  • Preadipocyte Differentiation Medium
  • Adipocyte Nutrition Medium
• 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
• Is it normal that CD34+ progenitor cells attach to tissue culture plastic after a week-long culture in HPC Expansion Medium XF (C-28021)?

  The cells sink down to the bottom of the culture vessel but don’t really attach. They retain a roundish morphology and can be rinsed off with culture medium easily.

  Related Links and Documents

  • Human CD34+ Progenitor Cells
  • Hematopoietic Progenitor Expansion Medium XF
• At what passage are PromoCell Human Blood Cells upon arrival?

  PromoCell Blood and Blood Progenitor Cells are cryopreserved directly after isolation (= P0). They haven’t been in culture before freezing.

  Related Links and Documents

  • hCD34+
  • hMoCD14+
  • hMNC
• 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
• 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
• What happens if I grow CD34+ or CD133+ progenitors in Hematopoietic Progenitor Cell Expansion Medium XF (C-28021) without Cytokine Mix E (C-39890)?

  The cells will die. Hematopoietic Progenitor Cell Expansion Medium XF always must be supplemented with Cytokine Mix E or an appropriate cocktail of cytokines.

  Related Links and Documents

  • Human CD34+ Progenitor Cells
  • Hematopoietic Progenitor Cell Expansion Medium XF
• 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 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
• 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
• 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
• How do I best isolate RNA from PromoCell cell pellets (C-14**)?

  There are two options for isolating RNA from cells stored in RNAlater Solution:

  1) The solution is removed from the cells prior to extraction by centrifugation at 5,000 x g for 10 minutes at 4°C.
  Note: Because of the density of RNAlater® solution, greater centrifugal forces are required to spin down the cells.

  2) If no pellet is visible after centrifugation, RNA can also be purified directly from the RNAlater® solution. This can be done by adding 2 ml of 10x lysis buffer, and proceeding normally.

   

  Related Links and Documents

  • Cell pellets
• 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
• 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

  • hMSC
  • Mesenchymal Stem Cell Media
• 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
• 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.

  Related Links and Documents

  • hMSC
  • Mesenchymal Stem Cell Media
• Can I expand the human Mesenchymal Stem Cells prior to differentiating them?

  These cells are frozen at the end of 2^nd culture. Thawing and seeding results in passage 2 (3^rd culture). We recommend that they be used for differentiation experiments not later than passage 5.
  The differentiation potential of hMSC in vitro is reduced with ongoing population doublings, meaning the earlier differentiation is induced, the higher the differentiation rates.

  Related Links and Documents

  • hMSC
• What is the expected differentiation rate when using PromoCell Mesenchymal Stem Cell Osteogenic Differentiation Medium with hMSC-BM?

  The differentiation rate into the osteogenic lineage is 70-100%.

  Related Links and Documents

  • hMSC-BM
  • MSC Osteogenic Differentiation Medium
  • Application Note: Osteogenic Differentiation and Analysis of MSC
• 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 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

  • HCH
  • Chondrocyte Growth Medium
  • Chondrogenic Differentiation and Analysis of MSC
• 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
• At what passage are PromoCell Mesenchymal Stem Cells / Pericytes upon arrival?

  Our Human Mesenchymal Stem Cells and Human Pericytes are cryopreserved at the end of secondary culture (P1). After thawing, they are in P2.

  Related Links and Documents

  • Human Pericytes
  • Human Mesenchymal Stem Cells
• How big is the portion of MSC that can be differentiated into neuronal lineages?

  Usually 90-100% of the hMSC show a neuronal morphology after differentiation with our Mesenchymal Stem Cell Neurogenic Differentiation Medium (C-28015). 60-80% of them are positive for nissl bodies after a nissl stain. However, the differentiation capacity depends on the origin of the cells and the number of population doublings they have undergone.

  Related Links and Documents

  • Instruction Manual: Mesenchymal Stem Cells
  • Instruction Manual: MSC Media
  • Application Note: Neurogenic Differentiation and Analysis of MSC
• 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
• After differentiation of human MSC into mature adipocytes – what medium do I have to use to culture those cells?

  Differentiation of hMSC into mature adipocytes takes approx. 2 weeks. You can keep the adipocytes for up to 3 weeks in the MSC Adipogenic Differentiation Medium. After 3 weeks we recommend to switch to PromoCell’s Adipocyte Nutrition Medium (C-27438).

  Related Links and Documents

  • Mesenchymal Stem Cells
  • MSC Adipogenic Differentiation Medium 2
  • Adipocyte Nutrition Medium
  • Application Note: Adipogenic Differentiation and Analysis of MSC
• How long can I keep neuronally differentiated MSC in culture?

  Changing the medium every 2-3 days, the neuronally differentiated MSC can be kept in culture for up to 2 weeks.

  Related Links and Documents

  • Instruction Manual: Mesenchymal Stem Cells
  • Instruction Manual: MSC Media
  • Application Note: Neurogenic Differentiation and Analysis of MSC
• I would like to know how homogenous the MSC population is and if all cells become osteoblasts when grown in MSC Osteogenic Differentiation Medium (C-28013).

  Human Mesenchymal Stem Cells have retained the ability to differentiate into a variety of cell types including fat cells, chondrocytes, and osteoblasts. PromoCell supply hMSC from 3 different tissues: bone marrow (hMSC-BM; C-12974), umbilical cord matrix (hMSC-UC; C-12971), and adipose tissue (hMSC-AT; C-12977).

  • MSC-BM show very good differentiation into bone cells but also into chondrocytes and fat cells when the respective Differentiation Media is used.
  • MSC-UC have a high potential to differentiate into chondrocytes but only weak potential for fat or bone cell differentiation.
  • In contrast, MSC-AT differentiate very well into fat and bone cells but only moderately into chondrocytes.

  In other words, to obtain a high percentage of bone cells, MSC-BM or MSC-AT are the cells of choice. There are of course lot-to-lot variations and the differentiation will decrease from passage to passage. If you need MSCs with a particularly high osteoblast differentiation capacity, you can call our Technical Customer Service before placing your order so that we can select an appropriate cell lot for you.

  Related Links and Documents

  • hMSC
  • MSC Differentiation Media
  • Application Note: Osteogenic Differentiation and Analysis of MSC
• What population doubling times can I expect when growing PromoCell’s Mesenchymal Stem Cells? How long does it take from seeding to subculture?

  The population doubling times for PromoCell hMSC (hMSC-BM, hMSC-AT, hMSC-UC) are typically ≤ 30 hrs when using Mesenchymal Stem Cell Growth Medium 2 (C-28009).
  If you seed the hMSC at 4.000 cells/cm², it will take between 4-7 days until they reach subconfluency.

  Related Links and Documents

  • Mesenchymal Stem Cells
  • MSC Growth Medium 2
• The Certificate of Analysis of hMNC contains the percentages of the main cell types (lymphocytes, monocytes, …). How does PromoCell determine that these are monocytes?

  Our MNC lots are checked for the rate of lymphocytes, monocytes and granulocytes w/o staining by FSC/SSC using a flow cytometer. A sample plot is attached. For our purified CD14⁺ monocytes we additionally perform a CD14⁺ staining.

  Related Links and Documents

  • hMNC
  • CD14 monocytes
  • Mononuclear cells_FACS
• 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
• 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
• 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
• Is there is a difference in the percentage of differentiated DCs depending on whether we start with cryopreserved or fresh isolated monocytes?

  There is no significant difference in the percentage of cells that will differentiate into Dendritic Cells. But when using cryopreserved cells, the initial cell loss will be higher compared to when fresh cells are used. i.e. the final number of differentiated cells that can be expected will be higher with fresh cells as a starting material due to lower cell death rate.

  Related Links and Documents

  • DC Generation Media
  • Generation of monocyte derived Dendritic Cells
• We use Macrophage Generation Media from PromoCell to get differentiated macrophages from peripheral blood monocytes. The monocytes are isolated by culturing PBMCs in Monocyte Attachment Media (also from PromoCell) for 1.5 h. Would it be ok if the attachment step is done overnight, since we receive the blood later in the day which makes it difficult to process on the same day?

  It is not recommended to leave the blood cells in the Monocyte Attachment Medium for longer than 1.5-2 hrs. The medium was developed for (short-term) attachment of the monocytes and does not provide nutrients for a longer time period. Leaving the cells in Monocyte Attachment Medium for a longer time or even overnight will induce apoptosis and lead to the loss of the cells.
  If necessary, you can reduce the incubation time to 1 hr. In this case, it is advisable to equilibrate the media in the incubator before so that you can immediately and directly add the appropriate amount of PBMC suspension.

  Related Links and Documents

  • Application Note: Differentiation of M1-or M2-Macrophages from PBMC/Monocytes
  • Monocyte Attachment Medium