Hej, I want to activate neutrophils and collect their released granular content. I would like to do it without any biological (LPS etc.) or chemical (PMA etc.) stimuli.
I tried harsh pipetting and vortexing of the cells, with a following incubation of 15min, then spin to collect protein content. However, the yield was fairly low.
Is there a more efficient way? Maybe longer incubation time? I would like to avoid total lysis of the cells, but maybe I triggered that already with my treatment?
Any suggestions are highly appreciated!!!
I have several suggestion:
You could trying thermal shock (45 to 1 to 45 celcius as fast as you can) or osmotic shock (distilled water for a minute). You can try to place your neutrophils in starvation for some times (i suggest 12 to 24h given the short half life of neutrophils, longer would lead to mainly apoptosis residues).
You could try physical membrane permeation, without lysis. maybe with a low intensity sonication or low voltage electroporation (pretty much as they do in molecular biology, but without inserting a plasmid or anything). It may works since it would allows a greater entry of calcium in the cell, which play roles in degranulation.