Hi Martine, First of all I would always titrate both the incubation and the concentration of Hoechst. We have found that it can vary with cell type even if they seem to come from a similar source. Also the cell concentration should be rigourously controlled and kept consistent. Although you dont mention it, I assume at the end of the Hoechst incubation, you are washing the cells before adding PI? It sounds like the level of Hoechst you are using may be cytotoxic if the death rate increases with time. You may be able to reduce it. We have seen what appear to be SP cells after short incubations but because they are not ablated by verapamil or similar inhibitor we cant be certain - this again is why a time course is vital because at some point (if you are lucky) you will get an SP that disappears in the verapamil tube. Ger van den Engh recently published a paper (Cytometry. 2004 Sep;61A(1):18-25) that explores the relationship of Hoechst and DNA which is quite enlightening! Good luck! Derek >We have been successful at isolating a "SP" population of epidermal >cells (low Hoechst 33342 and PI content) that we called Epidermal >Progenitor Cells. These cells are sensitive to Verapamil 50 uM. All >these experiments were done with neonatal cells. > >We are now trying to isolate the same population of cells from adult >skin. Our protocol calls for 90 min at 37 degrees in Hoechst, then >resuspend in PI and sort. When we do that, we are able to select a >population of cells that are verapamil sensitive. However, we have a >huge percentage of dead cells within this population (80%). We have >reduced the incubation time in the Hoechst to 30 min, and have seen >a major improvement in the death rate (50%). However, after 30 min, >they are not verapamil sensitive. The question is: can the verapamil >be effective in 30 min, or not? Can the Hoechst be effective in 30 >min or not? -- *************************************************************** Derek Davies Voice: (44) 020 7269 3394 FACS Laboratory, FAX: (44) 020 7269 3479 London Research Institute, e_mail: derek.davies@cancer.org.uk Cancer Research UK mobile: 07790 604112 44 Lincolns Inn Fields, London, UK. Web Page: http://science.cancerresearchuk.org/sci/facs/ In tenebris lux ***************************************************************Received on Fri Dec 17 12:38:00 2004
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