By Zbigniew Darzynkiewicz; Harry A Crissman
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Additional info for Methods in cell biology. / Volume 33, Flow cytometry
Wallen et al. (1983) modified the technique slightly using Hoechst 33342 (H0342) and PI. Hoechst stains all of the cells (live and dead), while PI stains only the dead cells. Hoechst fluorescence is quenched in the dead cells by PI, resulting in an increase in red fluorescence from PI. The resulting bivariate histograms show the equivalent of two single-parameter DNA histograms; one with high blue-low red fluorescence (live) and one with high red-low blue fluorescence (dead). , 1988). The method for H0342-PI staining in 25% ethanol will be described.
A) and (B) Control HEP-2 cells in exponential growth in v i m . The live (GIL, SL,G2+ M,) and dead (GID, S , , G, + MD) cells are labeled in panel (A). (C) and (D) HEP-2 cells after freeze-thawing twice (45% dead by both trypan blue and H0342-PI). 3. PHASE-SPECIFIC ANALYSIS OF VIABILITY 23 The addition of 25% ethanol after PI staining serves three purposes. First, the cells are permeabilized, facilitating H0342 staining without incubation at 37°C. Second, fixation in ethanol results in prolonged stability of the staining over 72 hours.
All of this is prerequisite to any investigation of the effects of biologic, chemical, and/or physical agents on cell MP. E. Pitfalls and Cautions Addition of any substantial amount of protein to a cell suspension that has been equilibrated with dye in a protein-free medium will decrease the dye concentration in cells, because the dye will bind to the protein in solution. , 1-10% albumin) when trying to 4. CELL MEMBRANE POTENTIAL ANALYSIS 33 determine effectsof adding specificproteins to cells.
Methods in cell biology. / Volume 33, Flow cytometry by Zbigniew Darzynkiewicz; Harry A Crissman