Hi Sally, In our hands, CSFE tends to bit leak from the cells, and the dye contaminated other cells adjacent to it. We use PKH26 from sigma with very good results. This dye is 'Locked' into the membrane of the cells so no dye leakage. The dye is exited at 488 nm and we collect the emission through a PE filter around 585nm. You can use this dye with other fluorochromes e.g. 7AAD, FITC etc. Hope this helps. Andy. >Dear Flow Folks, > >We are attempting to use CFSE to monitor in vitro MLR?s for a study of >alloregulatory cells in mice. Our model involves a relatively weak >response (single class plus minor antigens). > >Our responder cells are labelled with CFSE and, when harvested, stained >with either CD4- or CD8-Tricolor (Fl3). We take great care with >compensation, as the bright CFSE fluorescence spills into Fl2 and Fl3. We >live gate on lymphocytes/blasts using FSC/SSC. > >Our problem is with autofluorescence of the cells that are not labelled >with CFSE, stimulator cells, and particularly, the cells we are adding as >co-cultured cells: responder strain regulatory cells or responder strain >naïve cells (controls). The co-cultured cells are added as unfractionated >or fractionated splenocytes. > >The autofluorescence of the non-CFSE labelled cells increases during 72 hr >in culture until it spans 2 decades on the Fl1 log scale. The brighter >autofluorescence thus interferes with measuring the number of responder >cells that have undergone 4 or 5 to 8 divisions and are then CFSE dim. >Thus the usefulness of the CFSE measurement is severely limited. > >Our responder cells are washed well before culture and reutilization of >the CFSE label does not appear to be a problem. > >In contrast to the in vitro MLR, in vivo MLRs performed with CFSE give us >beautiful data (but unfortunately are not suitable for our regulatory cell >study). > >We would be grateful for any information or thoughts on this problem you >may have. > >Many thanks, > >Sally De Fazio >Albany College of Pharmacy & >Northeastern University -- Andy Riddell Flow and Imaging Lab CIMR Hills Road Cambridge UK tel: (0)1223 762597 fax: (0)1223 336900 email: ar289@cam.ac.uk
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