Technical Library

Recent Entries in Technical Library

• 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