Technical library

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.

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.

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.