| | Re: Cd11b detection
hm, I only used the MACS for a couple of time because I needed more PMN than I could isolate with this system. But according to the literature, your neutrophils should be at a very low activation level after this isolation procedure (after all, there are no long centrifugation steps as in Ficoll-isolation etc. ). This shouldn´t be a problem for you...
But keep in mind that pmn are very delicate cells, and unfortunately, they don´t like long incubation. Take a look at Wolach et al, 2007 (PMID: 17379064), they got only 26% living PMN after 16-18h incubation (w/o growth factors). So I could imagine that at least for your samples older 4-6h, your inconsistent results might be, at least in part, due to unspecific binding of your AB to dead/dying PMN...
I would run an apoptosis assay on all your timepoints, and fix your cells in 2% PFA directly after harvesting (altough you keep them on ice, my experience is that CD11b still changes...). CAVE: at least for Annexin-apoptosis-assays, do not fix these cells! (Annexin sort of tests your pmn membranes integrity, and you will get a lot of false-positives after fixation!)
hope that helps