Joan Kalnitsky writes- > I have a client who would like to sort potato protoplasts. We are >wondering if anyone out there does this and if so if anyone has any input >into whether Hoechts or DAPI would be a better choice. Do both of these >dyes work without affecting the viability of the cells. They definately >want live cells when we are done. > What about 7-AAD. We currently run a lot of 7-AAD in our lab > looking at >apoptosis. I know it is used as a DNA dye. Does it provide ploidy peaks? >If so does anyone have any input as to whether this dye would be a good >choice for this undertaking. > One other question while i'm at it. For the DAPI and Hoechts, what >filters would you recomend using to measure emission? In the literature >they had, there were conflicting reports of wavelengths. Hoechst 33342 definitely gets into live mammalian cells better than either DAPI or 7-AAD, to which the membranes of viable cells are normally impermeable. I would guess that your protoplasts would take up the Hoechst dye; if they took up 7-AAD, I'd be concerned about their viability, and I wouldn't expect them to take up DAPI, although some viable bacteria seem to. In fact, it might be advisable to run Hoechst 33342 with 7-AAD, and only sort the cells which don't take up 7-AAD. If you have more than 5 mW of UV excitation, it doesn't much matter what filter you use for Hoechst dye. I generally use a 450 nm/ 20-30 nm bandwidth filter to keep the 488 nm in the second beam out of the Hoechst channel. -Howard
This archive was generated by hypermail 2b29 : Sat Mar 10 2001 - 19:31:26 EST