Ethanol-fixation of Samples for Long-term Storage and Subsequent DNA Staining

I.            Materials

70% Ethanol at – 20^oC

DNA Staining Buffer:

Sodium citrate    0.25g
Triton–x 100    0.75ml
Propidium iodide   0.025g
Ribonuclease A      0.005g
Distilled water      250 ml

II.            Procedure

1.      Place 1x10⁶ cells from each sample into a polypropylene tube and centrifuge at 250 x g for 5 min. 

2.      Remove the supernatant as completely as possible without disturbing the pellet and add  1 ml of –20^oC 70% EtOH dropwise to the cell pellet while vortexing.

3.      Keep cells at -20^oC until the day of DNA staining (cells can be stored for several weeks at -20^oC).

4.      On the day of DNA staining, take samples out of the freezer and spin them down by centrifugation at 250 x g for 5 min.  Remove the supernatant as completely as possible without disturbing the cell pellet.

5.      Add 1 ml of DNA staining buffer to the cell pellet and vortex gently and briefly.  Keep cells for 15 min in the staining solution before acquisition on the flow cytometer.

Commercial sources:

Sodium citrate              Cat# C7254 Sigma, St. Louis, MO
Triton-x 100                 Cat# x-100                              "
Ribonuclease A            Cat# R4875                       "
Propidium iodide   Cat# 537059            Calbiochem, San Diego, CA