From: Steve Perfetto (sperfetto@hiv.hjf.org)
Date: Thu Jun 18 1998 - 13:51:47 EST
Derek, This procedure has been very successful in our hands and we now have combined it with Apoptosis and CD25-PE. Please note that these are only possible with the use of a green HeNe and gated amp because the CFSE activation contaminates most of your PMT's. We run a 96 hour time course under many conditions but mostly using activated T cells (see protocol below). However, it is unlikely that the Jurkat's are giving you this problem and I suspect it is your concentration. Under the microscope and in the flow these cells are very bright. Our standard dose is 6µM in Hank's (with calcium and magnesium ions) which is added to equal volumes of a cell suspension at 25E6 per ml. 8 minutes at RT , mixing only occasionally. Wash 1X in Hank's followed by 3X in culture media. Set in incubator and withdraw at study intervals. REF:JIM, 171 (1994) 131-137; B.Lyons and C.Parish. I hope this helps. Stephen P. Perfetto, MS.,MT. (ASCP) Walter Reed Army Institute of Research Department of Molecular Diagnostics and Pathogenesis 1600 East Gude Drive Rockville, MD. 20850 _______________________________________________________________________________ Subject: CFSE and cell division From: Derek Davies <daviesd2@icrf.icnet.uk> at Internet_Gateway Date: 6/17/98 5:31 PM Dear World, We are venturing into the world of following cell divisions using the CFSE dye from Molecular Probes. The first attempt appears to have been an abject failure. Cells (Jurkat J6) have been loaded with CFSE at 10nM for 10 mins at 37C as per a published protocol, but there is no fluorescence at all after the loading period. The stock CFSE is stored in DMSO at -20C at a 5mM concentration. Obviously we may not have the conditions right and the users my are now varying the "staining" times and dye concentrations but we were wondering whether anyone had practical experience of this - a quick Medline search didnt yield too many references. Thanks in advance, Derek **************************************************************************** * Derek Davies Voice: (44) 0171 269 3394 * * FACS Laboratory, FAX: (44) 0171 269 3100 * * Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk * * London, UK * * * * Web Page: http://www.icnet.uk/axp/facs/davies/index.html * ****************************************************************************
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