Hello,
A few weeks ago, someone wrote in asking about how to detach
monocytes for use in flow, as they stick to everything. I am
experiencing a similar problem.
Here is my protocol:
I stimulate PBMC in polypropylene tubes for 3 and 24 hours. Afterwards,
I wash the cells in 1 x PBS, 1% FCS, 0.1% sodium azide and 0.4% EDTA
twice, stain for surface molecules, wash twice more in the same wash,
then I fix and permeabilize the cells with PharMingens Cytofix/Cytoperm
reagent (I don't think this contains any EDTA). I was twice with the
permeabilization reagent, stain for intracellular cytokines, wash twice
more in the permeabilization reagent and resuspend in my original wash
solution. All the staining and washing occurs in eppendorf tubes, and
after I resuspend the PBMC, I transfer the cells to flow tubes. I vortex
strongly between each wash and by 3hrs I typically lose 50% of the
monocytes in my stimulated groups compared to unstimulated and by 24
hrs, my unstimulated groups have lost about 80% and stimulated groups
close to 90%. The monocytes I do have, have a much larger side scatter
profile when compared to 3hrs. Any help would be greatly appreciated.
Slava Epelman
sepelman@ucalgary.ca
University of Calgary
Dept of MID
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