Hi All,
I am trying to optimize a protocol for neutrophil isolation by FACS.
My question is how many cells should I expect from 2 femurs?
This protocol is based on a first step of neutrophil enrichment by negative magnetic depletion of CD19 and Ter119.
And then staining and gating of CD11b and Ly6G double positives (with a B220 to check the efficiency of negative depletion).
I am getting a purity of 100% but only 60 000 cells /mouse.
Thanks,
Dario
Hi,
I don't have any experience with enriching neutrophils, but how many cells are you getting fromt he two femurs and from which strain and how old are your mice?
We usually get 20-25x10^6 cells from 8-10 week old mice, quite a bit less if the mice are younger.
Just to check if your isolation is working fine.
I think you may want to check your isolation procedure - maybe also check the cells left behind in magnets (by flushing columns).
I regularly get around 10^7 granulocytes by density gradient separation (~85% Ly6G positive by flow) from 2 femurs and 2 tibias (although majority of cells are in femurs).
If your neutrophils are activated you may find them bound to red cells (and hence your Ter119 beads) - if that's the case you might try adding DNAse (or similar), which can reduce this.
Thank you guys. I improved the isolation by flushing the femurs and tibias more carefully and reduced the amount of magnetic beads. Got 4x10^6 cells from one mouse, 99% Ly6G/CD11b positives. This is enough but I will try further the DNAse suggestion.
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