HomeLive Go cell cycle analysis with Hoechst 33342 and Pyronin Y
Flow cytometry may be used to determine the level of senescence in a cell population. Go and G1 can be separated by use of Pyronin Y (Sigma) which preferentially binds to RNA and Hoechst 33342 (Sigma) which binds to A-T base pairs. Cells were loaded with Hoechst 33342 at 10 mg/ml and Pyronin Y at 0.5 mg/ml by incubating for 45 mins at 37C. Pyronin Y is excited at 488 nm and emits at 575 nm, while Hoechst 33342 is excited UV lasers (350-360 nm), near UV line (375 nm) or violet diode (405 nm).
Examples of fibroblasts and Jurkat T cells stained with Hoechst 33342 and Pyronin Y show the presence of Go cells with little or no RNA present whereas the G1 cells show high level expression of RNA. The small amount of RNA present in Go cells was so low using the same PMT settings on a Zeiss confocal no RNA was visible. Therefore the Go cell imaging was carried out at 250 volts higher than the G1 cycling cells. The confocal imaging of Pyronin Y in Go senescent and G1 cycling cells clearly shows a large difference RNA content of these cells in the two stages of the cell cycle.
