Technical library

Osteogenic differentiation of hMSCs with PromoCell MSC Osteogenic Differentiation Medium takes ∼14 days (please view the respective Application Note or Product Manual for a detailed protocol).

Use collagen- or 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.

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

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.

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.

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.

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%.

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.

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

The recommended seeding density with 100,000 cells per cm²is 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.

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