Tara, I am one of those people who use EDTA often to release cells from plastic/glass vessels but cell to cell sticking usually is not helped much by EDTA. It would not hurt to try adding some at a comcentration of 0.6mM but I think the follow tips will help you more. First the ethanol needs to be ICE COLD. I usually place mine in a -70C for at least one hr or -20C over night before the fixation. Your cells need to be in the best single cell (clump free) suspension as possible. When you start to fix your cells, vortex the ICE COLD ethanol adding the cells sowly while vortexing. Then I would add either 1% of a protein like BSA or FBS. Most of the cell to cell clumping after fixation is due to the attraction of the cell surfaces to each other and once they make contact they get tangled in each others cell surface proteins even though they are fixed. The 1% protein will help coat the fixed surface of the cells preventing them from sticking together. GOOD LUCK Tim Chance CDC Atlanta,GA -----Original Message----- From: Tara McDonald [mailto:t.mcdonald@centenary.usyd.edu.AU] Sent: Wednesday, June 12, 2002 2:36 AM To: cyto-inbox Subject: EDTA to help unstick sticky cells 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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