I have done considerable 3 color phenotyping off of 488 using and used UV to attain a cell cycle by DAPI staining. My observations pretty much parallel the postings so far. A titer of DAPI concentration for DNA staining vs DNA fluorescence profile analyzed by Multicycle generally demonstrates DAPI concentration in the 1.5-2.5 ug/ml gives the best cell cycle result but as described, the fluorescent noise in the FITC channel is obnoxious! I use a Vantage and over the years have discovered part of the problem is optical alignment. The further away from minimum width laser focus, the worse the noise. I have been told it may have to do with light piping up the sheath or sample column. Howard Shapiro's comment makes alot of practicle sense. Fresh cells I've seen stained with Hoechst 33342 and GFP show no DNA dye noise in the green channel. Excellent cell cycle! The GFP signal was > 2X that of any FITC-antibody staining I've seen. Two secrets here--fresh cells and the low gain setting for detection of GFP made HO noise in green fluorescence unnoticable. Cells fixed in 1.25% formaldehyde for a short period of time and held in buffered saline+2% FBS + azide seems to have much less DAPI staining noise in the FITC channel than cells fixed in 2% formaldehyde overnight, washed, and stained immediately. In the first case cells may be stained overnight in DAPI and in the second case, usually for less than 3 hours. Last observation. DAPI staining in the presence of protein for either ethanol or formaldehyde fixed typically reduces DNA staining noise in the green channel. However, the degree of noise reduction is irritatingly variable. S O o o--Will someone volunteer accurate infomation on: 1) Optical alignment and its influence on Hoechst/DAPI fluorescence in proper and improper channels. 2) Continue the discussion on Ho/DAPI dyes and fixation especially if you've tried the glycine block idea. What is the result here. 3) In the last observation, why is the result variable? DAPI stock and FBS stock remain constant along with the HBSS salt source, Milli-Q water souce. pH variablity 7.2-7.4 with preparation & age. Louis King kingl@pilot.msu.edu
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