From: Andrea Dewar (andrea.dewar@imvs.sa.gov.au)
Date: Wed May 14 2003 - 03:00:36 EST
Hi there, I'm growing human macrophages in 24 well plates from PB-derived monocytes, by stimulating the monocytes with either GM-CSF or M-CSF for 1 week. I am then performing various functional assays with these cells, such as antigen uptake assays and a measure of the ability of these cells to produce proinflammatory cytokines. Initially I was using ELISA to measure the levels of cytokines, but am now wanting to look on a per-cell basis using flow cytometry (and intracellular staining). The problem I am having is that my macrophages don't want to let go of the culture plate. This causes enormous problems for my intracellular cytokine assay, as at the moment I am rupturing the cells; as a consequence the cells release all their cytokines and look very shabby on FS and SS plots. Also, if I use 7AAD staining to gate out dead cells, most of my cells take up 7AAD due to rupturing, when prior to harvest, the cells were 90% viable (this bad 7AAD staining is on samples that have not been fixed/permeabilised). Can anyone help me with techniques for getting the macrophages off the culture plates? I have tried extensive trypsin/EDTA treatment (leaving the cells for up to 2 hours does nothing), sitting the cells on ice, and a combination of trypsin/EDTA, collagenase and dispase, to no avail. Gentle scaping does get the cells off, but they don't like that at all, and as a consequence rupture. Any help would be greatly appreciated! Many thanks, Andrea Andrea Dewar PhD Student Leukaemia Research Laboratory Department of Haematology Institute of Medical and Veterinary Science Adelaide South Australia 5000 AUSTRALIA Tel. +61 8222 3498 or +61 8222 3071 Fax. +61 8 8222 3139
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