Preparation of Yeasts for FACS

							FACS with Propidium Iodide


  * spin down 100 - 1000 µl cells (depending on cell density)
  * resuspend in 1 ml 70 % Ethanol and rotate for 30 min at room temperature (up to
several days)
  * or: add 400 µl cells directly to 850 µl 100 % Ethanol
  * pellet cells
  * wash 2 x with 1 ml 50 mM Tris pH 7.8
  * resuspend cells in 500 µl 50 mM Tris
  * add 10 µl RNase A (10 mg/ml), incubate 1- 4 hrs at 37 °C
  * pellet cells
  * resuspend in 500 µl FACS-buffer
  * add 15 µl propidium iodide (1 mg/ml)
  * samples can be analysed immediately or stored at 4 °C
  * before analysis sonicate for 10 sec. (setting low - medium)


Buffers:

FACS-buffer:
200 mM Tris/HCl pH 7.5; 200 mM NaCl; 78 mM MgCl2.