Silver: PI FACS

From OpenWetWare

Jump to: navigation, search

Back to Silver Lab

Back to Protocols


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:

  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

Day 2:

  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
    • (1.5 ml 10 mg/ml RNAse A + 13.5 ml 50 mM Tris-HCl pH 8.0)

Day 3:

  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

Personal tools