Good afternoon.
I have some concerns regarding my gating strategy for a phagocytic assay.
Here is the experimental setup :
I incubated mouse bone marrow derived macrophages (BMDM) with B16F10-GFP murine cancer cells for 2 hours at two different ratios : 1:2 (excess of cancer cells) and 4:1 (excess of BMDM).
After two hours, I stained BMDM with a CD11b-PE-Cy7 antibody in cold MACS buffer (contains PBS, BSA and EDTA ; I guess EDTA is 2 mM) for 15 minutes in the dark in the fridge.
Then I wash the cells with MACS buffer (it means I fill the tube with MACS buffer, spin it at 300 g for 5 minutes at 4 degrees, remove the supernatant and resuspend the cell pellet in 400 uL of MACS buffer) and go to acquire the cells on a flow cytometer.
The goal of the assay is to figure what is the percentage of CD11b+ BMDM which engulfed cancer cells (so these CD11b+ BMDM should be positive for GFP as well).
My concern is how to gate regarding singlets / doublets.
Indeed on the 1st attached picture, you can see that if I use all the cells (without removing doublets), about 25 % of the BMDM are GFP + at a 2:1 ratio.
But if I only gate on the singlets, I cannot see the same percentages anymore (2nd picture).
I wanted to remove the doublets because sometimes what can happens is that BMDM and cancer cells are engaged in what we called a conjugate, with an immunological synapse between the two cell types. This means that the cancer cells has not been engulfed yet.
The problem is that at the same time, BMDM which engulfed cancer cells might have become bigger, so they might have a higher FSC-W and FSC-H and thus might be discarded when only keeping the singlets.
However with a concentration of 2 mM EDTA and an incubation of 15 minutes, I would assume that any conjugates should be disrupted right ? (usually conjugates formation is highly dependent on calcium, which is chelated in presence of EDTA). While at the same time, truly engulfed cancer cells by BMDM should not be affected by the presence of EDTA.
I just would to see how you usually gate your cells in similar assays and would be happy to have any feedbacks related to it.
Thanks a lot for your help.
I wish you a nice day.
Best regards
Jérôme
https://www.researchgate.net/figure/Gating-strategy-for-in-vivo-phagocytosis-Gating-strategy-for-in-vivo-TAM-phagocytosis-of_fig8_334817436
Cancer can be interpreted as unpacking of prootozoan intentions by multicellular's cell as a reponce to stress factors. Survival programs are related mainly with HSPs and block apoptosis programs that is fatal.
The live/dead staining is essential step, and its gating should be performed before the FACS analysis.
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