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 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%.

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.

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.

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 cm 2 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.

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.

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.

Has PromoCell done any tests with MSC Adipogenic Differentiation Medium 2 without fibronectin? Is fibronectin coating really necessary?

The MSCs attach even without fibronectin coating, but the risk of detachment during differentiation is much higher. We therefore strongly recommend using fibronectin- or vitronectin-coated TC vessels.

Technical library - RESOURCES

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.