Flow cytometry in yeast (Propidium Iodide)

- Add 700 μl 100% ethanol to 300 μl culture (1X107 cells). Vortex 3 seconds.

Optional: Store at 4°C. Can be stored for up to 1 years before staining.

- Vortex until dissolve the pellet completely and take 100-200 μl.

- Centrifuge cells at 13000 rpm, 5 min, 4°C.

- Resuspend pellet in 0.5 ml 50 mM Sodium Citrate and 0.2 μg/ml RNAse. Incubate 2 hours or ON at 37°C.

- Centrifuge cells at 13000 rpm, 5 min, 4°C.

- Resuspend pellet in 0.5 ml 50 mM Sodium Citrate, 50 mM HCl and 5 mg/ml pepsine. Incubate 30 min at 37°C.

- Centrifuge cells at 13000 rpm, 5 min, 4°C.

- Resuspend pellet in 1 ml 50 mM Sodium Citrate and 2 μg/ml Propidium Iodide.

- Transfer samples to FACS tube and sonicate 10 seconds.

 

Acquire data on flow cytometer within 48 hours (but might last up to 2 weeks).

 
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