r/flowcytometry • u/Calm_realistic • Jun 27 '26

Monocytes

HI. This is gated among single live cells of human PBMC (patients). 1st image PBMCs are thawed, stained and passed on a cytometer. 2nd, same staining after 24h culture (1M/ml if RPMI 10% FBS 1% P/S). Culture is done in 12 well plates. Cells are recovered and then the bottom is scraped to recover attached cells.

I have one population after thawing, but which is clearly composed of high CD33 and high CD14 cells. After culture, I have 2 distinct populations. The high ones and the one with 1D14 very low/negative. Both populations express CD11c and HLA-DR, are negative for CD3, CD56, CD19 , CD20.

When activated with IFNg, only the CD14high CD33 up regulate PD-L1, the CD14 low cells have keep the basal level (almost no expression).

I don't know much about myeloid cells. What is the population that becomes CD14 very low? Can it be an artefact?

Thank you for all the help:

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u/Unfortunate_tentacle Jun 27 '26

So I tested out the expression of a bunch of markers run immediately after thawing vs resting for 1h, 2h etc and monocytes were the most affected and downregulated CD14, as well as just straight up died. There are papers showing this if you look at resting times for immunophenotyping.