Does FBS from Australia and South America have a significant impact on the differentiation of primary macrophages?

Primary macrophages are also sensitive to complement activity, and the composition of complement in South American serum may differ from that in Australia. Do not heat inactivate immediately, as heat inactivation can result in the loss of some growth factors and adhesion related proteins, which can be difficult to assess. You can start with two control groups: non inactivated and inactivated. Another thing to note is that there is greater fluctuation between batches in South America, and the first batch being OK does not mean that the subsequent batches will also be OK. At least two batch numbers should be tested. M-CSF concentration and differentiation time may need to be recalibrated and should not be based on the Australian system.

When Australian supply is tight, my strategy is "secure key items from Australia, expand with South America." If I must switch to South American sources, first sign a small-sample returnable/exchangeable agreement with the supplier and test 2-3 South American lot numbers. For flow cytometry, at minimum test F4/80, CD11b, CD80, CD86, CD206, and MHC-II; for qPCR, look at Tnf, Il6, Nos2, Arg1, and Mrc1. Pass them stably for 2-3 generations before drawing conclusions, and keep a control without the switched serum during this period; otherwise it is very difficult to determine whether differences come from the serum or from operational variability. Do not switch to large-volume purchasing just because the price is low.

The differences between Australia and South America cannot be simply based on their place of origin, but rather on endotoxins, hemoglobin, hormones, and complement components. Primary macrophages are highly sensitive to endotoxins, and some South American batches may have higher levels of endotoxins, which can easily activate M0 background. Before changing, the South American batch number COA should be obtained, and the endotoxin should preferably be ≤ 5 EU/mL. When verifying, do not only look at CD11b and F4/80, polarization markers such as iNOS, CD206, MHC-II should be included to determine whether they affect M1/M2 differentiation.

We conducted a same-period parallel comparison between Australian and South American lots. The same bone marrow batch was cultured in two dishes, and the differentiation rate of the South American lot BMDM indeed decreased, with M1/M2 markers also shifted. It is recommended not to switch directly; first perform a parallel experiment with a small sample: use flow cytometry to check the F4/80/CD11b double-positive ratio, and qPCR to measure Tnf, Il6, Nos2, Arg1, and Mrc1. The conclusion is that the full set of differentiation marker validation must be redone, and the original gating and thresholds cannot be directly applied. Keep the Australian lot for key experiments, and use the South American lot for prescreening or non-critical expansion.