Silver: PI FACS

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Materials

 * 100% ETOH
 * 50 mM Tris-HCl pH 8.0
 * RNAse A 10 mg/ml
 * 55 mM HCl containing 5 mg/ml Pepsin
 * Buffer A:
 * 10 ml 1M Tris-HCl pH 7.5 (200 mM Tris-HCl pH 7.5)
 * 2 ml   5M NaCl (200 mM NaCl)
 * 4 ml   1M MgCl2 (80 mM MgCl2)
 * Propidium Iodide stock (5 mg/ml)

Procedure
Day 1: Day 2: Day 3:
 * 1) 2.5 ml of 6 x 106 cells/ml into conical tubes containing 5 ml 100% ETOH
 * 2) Fix overnight on a rocker at the temperature the cells were originially grown
 * 1) Pellet and resuspend in 10 ml 50 mM Tris-HCl pH 8.0
 * 2) Put on ice for 1-2 h
 * 3) Sonicate at setting of ‘4’ for 20 seconds
 * 4) Pellet and resuspend in 0.8 ml of 50 mM Tris-HCl pH 8.0 with 1 mg/ml RNAse A, incubate overnight at 37°C
 * 5) * (1.5 ml 10 mg/ml RNAse A + 13.5 ml 50 mM Tris-HCl pH 8.0)
 * 1) Pellet and resuspend in 2 ml of 50 mM Tris-HCl containing 5 mg/ml pepsin; incubate for 30 min at 37°C
 * 2) Pellet and wash once with 2 ml Buffer A
 * 3) Pellet and resuspend in 450 µl Buffer A
 * 4) Add 50 ul propidium iodide stock (5mg/ml) to stain cells; final concentration of propidium iodide is 0.5 mg/ml (can be frozen and stored at -20°C at this point)

Transfer 100 µl cells to 400 ul of 50 mM Tris-HCl pH 8.0 immediately before reading in a FACS machine