Hi Douglas, I can recommend this paper "Regulation of imaginal disc cell size, cell number, and organ size by Drosophila class 1 (A) phosphoinositide 3-kinase and its adapter". Curr Biol. 1999 Sep23(18) 1019-29. Staining after trypsinisation doesn't work in our hands. The protocol she is using is correct. What improves the CV's is trypsinisation at slightly higher temperatures which in turn reduces trypsinsation times. However it's risky as the disc cells appear to be very fragile- the skill in obtaining the discs before trysinisation seems to play a role: e.g here 3 people doing a bulk preparation -the sample per person varies from good to very bad viability depending on who did the job. Our best CV's have been about 8%; day to day averages about 10%. If you are getting better than that please let me know. Same result using PI. I don't think you can apply the same criteria as for mammalian cells; the DNA profile is different as the cells have a very short S-phase and maybe this contributes to higher CV's. One of the drawbacks is the limited amount of sample so one doesn't have much leeway in trying to improve the proocol. As for the Hoechst it should be dissolved in water, and always a fresh aliquot. Ann At 13:03 31.10.01 -0500, you wrote: > >A collaborator is staining different fly parts (wings, imaginal disks, eyes, >etc) for cell cycle. >Flies have been modified with a bicistronic vector so that they can express >different proteins in tandem w/ GFP. >The current protocol she uses is to stain cells for 3 hours with 0.5ug/mL >Hoechst while the cells are being digested w/ trypsin during this same time >point. >Staining looks "okay", but the CVs are pretty wide - unacceptably wide, >actually. >We can't increase Hoechst concentration (toxic), and decreasing incubation >time doesn't seem to work either. The last hope is to stain after tissue >trypsinization/dissociation. > >So...does anyone know of ways to improve this? > >Thanks, >do > >----------------------------------------------------------- >Douglas P. Olson >Experimental Hematology/AIDS Research Center >Massachusetts General Hospital >Harvard Medical School >149 13th Street >Boston, MA 02129 >(617)724-2668 - Phone >(617)726-4691 - Fax >Dolson4@partners.org - Email >http://www.mgh.harvard.edu/aids > > >
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