Hi all, A researcher in my institute sorts ethanol fixed cells, subsequently stained with propidium iodide and FITC- or APC-conjugated antibody, for cell cycle analysis. At this stage there seems to be no alternative to the ethanol fixation for the particular protocol that he is using, so what we are 'stuck' with (pardon the pun) are sticky cells. He has a limited number of cells to sort and with the abort rate so high (can be up to 50-60% on a bad day) due to the stickiness we are losing a lot of cells and getting lower yields than is desirable. So far to minimise this I have suggested keeping cells on ice prior to and during sorting and pre-sort filtration through a 70um nylon filter to get rid of larger clumps. This has improved the situation only minimally. It has been suggested to me that EDTA can be used to deter the clumping but no details on the concentration etc. So my question is: has anyone out there used EDTA and if so what is the concentration you used and did it have any undesirable effects on particular stains/Ab binding/fluorochromes etc. All suggestions are greatly appreciated. Thanks in advance. Cheers, Tara. -- ******************************************************** Tara McDonald, BSc. (Hons.) Flow Cytometry Facility Centenary Institute of Cancer Medicine and Cell Biology Locked Bag No. 6 Newtown NSW 2042 AUSTRALIA Ph: +61 2 9565 6140 Email: t.mcdonald@centenary.usyd.edu.au ********************************************************
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