From: Geoffrey Osborne (Geoff.Osborne@anu.edu.au)
Date: Fri Jun 19 1998 - 06:44:30 EST
Hi Derek, Regarding your CFSE inquiry, I'd check the final concentration of CFSE that your users have. The often referenced paper by Lyons et al for work done here at JCSMR used a final concentration of *10uM*, not 10nM so you may want to check that. Also final concentration and incubation time are very much cell dependent, so for example fibroblasts need 5uM, 20 minutes incubation. I can't think what the conditions are for Jurkats at the moment, but I think someone here has done them. Sorry. Just another point worth noting, is that if you are dealing with populations of cells which are heterogenious with regard to size and phase of the cell cycle, then don't expect to see absolutely clear distinction following each cell division. Sometimes the total CFSE fluorescence of the population under study will decrease, and only small aberration in the edge of a histogram of CFSE fluorescence will indicate where some of the cells are synchronised. Hope this helps Geoff At 05:31 PM 6/17/98 +0100, you wrote: > > >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 * >**************************************************************************** > > > > ====================================================================== Geoffrey Osborne | ____ __ o Ahh! Flow Cytometry (FACS LAB) | __ `\ <,_ John Curtin School of Medical Research, | __ (*)/ (*) Australian National University, | ==============| CANBERRA, AUSTRALIA. | |--| Email: Geoff.Osborne@anu.edu.au | |--|... Phone: 61 2 6249 3694 | FAX: 61 2 6249 2595 | -----Surfing the Web?: Try http://jcsmr.anu.edu.au/facshome.html------ ======================================================================
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