I am doing the experiment for testing the percentage of immune cells in the peritoneal fluid cells(mouse), i repeated the experiment but each group of data from different mouse were different (the same condition for these mice) and also detect few NK cells and macrophages (i check the normal percentage of NK cells and macrophages should be 7% and 9% in my experiment).I collected the peritoneal fluid by adding 3ml FACS Buffer through Ip injection, cells were centrifuged for 5 minutes (500 G, 4°C), and then added Fc block before staining and staining within 6 hours. I use CD49b for NK cells, CD11b and F4/80 for macrophages, all the antibody is effective. Can you help me to find the problem of my experiment?
Hi Ya,
A few questions:
- How many cells/sample do you generally get over all? Of those, how many are CD11b+?
- Are you stimulating with a source of peritoneal inflammation before harvesting? The yield of macrophages without an eliciting agent can be relatively low (500,000 cells)
Macrophage Markers: Our lab has never had much luck with F4/80 in flow cytometry experiments, so I might recommend switching to CD68, which tends to work a little better.
NK Cell Markers: Resident NK cells of the peritoneum actually tend to be low expressors of CD49b, which may be part of the problem you're having. NK1.1 might be a better marker for characterization of these cells, see attached paper for antibodies and gating strategy: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3237802/
I suggest to use cell liability staining using dead cell markers to see how much your cells are live and dead at the same time. you need to optimize the concentration of your cell marker and the time you keep your cells in the FC block seems longer and such an extended time may decrease cell liability .
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