Technical library

PromoCell recommends using Human Serum AB, off‑the‑clot, for the generation of M0 macrophages.

Osteogenic differentiation of human mesenchymal stem cells (hMSC) using PromoCell MSC Osteogenic Differentiation Medium typically takes approximately 14 days.
A detailed, validated protocol is provided in the Product Manual and the corresponding Application Note.
 
Recommended culture conditions

• Use collagen‑ or fibronectin‑coated tissue culture plates.
• Change the medium every third day during differentiation.

Maintenance after differentiation
After approximately 2 weeks, when osteogenic differentiation is complete, the differentiated bone cells tend to detach from uncoated plastic surfaces.
For this reason:

• Functional assays and analyses should be performed promptly after differentiation.
• Cells should be maintained in MSC Osteogenic Differentiation Medium until the end of the experiment.

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.

In the PromoCell macrophage differentiation system, the polarization into M1 or M2 macrophages is completed within 7 days, which is consistent with reports in the literature and protocols used by other researchers.
 
Why the protocol spans 10 days
The PromoCell protocol includes an additional 2–3 days for optional macrophage activation. These extra days are intentionally included to improve overall culture performance.
Even if you aim to obtain non‑activated M1 or M2 macrophages, PromoCell does not recommend shortening the full 10‑day protocol, because:

• A medium change on days 8–10 promotes re‑attachment of previously floating cells
• This results in higher cell viability
• Overall cell yield is increased

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 of 100,000 cells/cm² for PromoCell M1 macrophages is required to achieve a confluent cell layer, as these cells do not proliferate after seeding.

Reducing the seeding density:
Yes. If a fully confluent monolayer is not essential for your assay, the seeding density can be reduced by a factor of 3 to 5.