Flow Cytometry Laboratory

Hoechst-Propidium iodide staining for apoptotic cells

1. Take cells in medium at a concentration no higher than 106/ml. Keep at 37°C until needed.

2. To 1 ml of cell suspension add 2 drops of propidium iodide (50 g/ml) and 100 l of Hoechst 33342 (10 g/ml). Leave for 4 minutes at room temperature and analyse by flow cytometry.

3. Flow cytometry: Dual laser. The primary laser at 488nm to excite the propidium iodide and the secondary laser at ultraviolet (351.1 - 363.8 nm) to excite the Hoechst. Both signals are logarithmically amplified with PI fluorescence being detected above 600 nm and Hoechst fluorescence collected between 390 and 480 nm.

The final concentration of Hoechst should be about 1g/ml. I have a stock of 1mg/ml from which the 10g/ml is made fresh before use. The influx of Hoechst into cells is time-dependent, the cells that stain in the first few minutes are the apoptotic (and dead) cells. If left long enough all cells will become Hoechst positive, so always keep a supply of unstained cells for emergencies. We are currently investigating the possibility of using Hoechst 33258 which takes longer to get into the apoptotic cells, but will not enter live cells to any great extent, so time will be less of a critical factor.

The apoptotic cells can be sorted so that they can be shown to be apoptotic either by looking for DNA-laddering, by electron microscopy or by acridine orange staining and fluorescence microscopy. The last is best as it requires the fewest cells. When sorting, remember to stain only small quantities of cells at a time.

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