Collecting samples and fixing cells in ethanol.
1) Chill a bottle of agd H2O in the frig and make sure there is a stock of 100% ethanol in the freezer.
2) Grow cells to desired density based on the experiment.
3) Prepare eppendorf tubes containing 10 μl of 10% NaN3 and keep them on ice.
4) Add 1 ml of cells to the tube and mix by vortexing. Incubate on ice while waiting to collect other samples.
5) Once all samples are collected, spin down at 5,000 x rpm for 5 min at 4 oC.
6) Aspirate the sup.
7) Add 1 ml of cold agd H2O and mix by vortexing.
8) Spin at 5,000 x rpm for 5 min at 4oC.
9) Aspirate the sup.
10) Add 300 μl of cold agd H2O.
11) While vortexing, add slowly 700 μl of cold 100% ethanol to fix cells. Adding ethanol slowly prevents excessive clumping of cells.
12) Vortex to mix and store the samples at 4oC until ready for flow cytometry preparation. Samples can be stored for months.
Flow cytometry with SYTOX Green
13) Transfer 500 μl of cells to new eppendorf tubes
14) Spin down at 4oC at 5,000 rpm for 5 minutes
15) Aspirate supernatant
16) Resuspend in 1 ml of 50 mM sodium citrate, pH 7.4
17) Vortex to mix
18) Spin down at 4oC at 5,000 rpm for 5 minutes
19) Resuspend in 1 ml of 50 mM sodium citrate, pH 7.4 containing 50 μg/ml RNase (made fresh from 10 mg/ml RNase stock and 50 mM sodium citrate buffer)
20) Vortex to mix
21) Incubate at 50-55oC for 1 hr
22) Add 50 μl of 20 mg/ml proteinase K and continue incubation for 1 hr
23) During the last incubation, prepare 1 mM SYTOX Green in 50 mM sodium citrate buffer (stock solution of SYTOX green is 5 mM, therefore 5000x)
24) Wipe outside of tubes dry. Spin down at 4oC at 5,000 rpm for 5 minutes
25) Add 1 ml of the SYTOX solution to cells
26) Vortex to mix and transfer to FACS tubes
27) Let cells incubate in the SYTOX solution for at least 0.5 hr. It’s OK to read the samples the next day. Store samples at 4oC
28) Sonicate cells using the probe sonicator in Biochem common room at setting 4, constant, 8 pulses of 1 second each pulse. Alternatively, use our water sonicator on HIGH for 1 min twice with 1 min cooling on ice between.
29) Transfer cells to BD FACS tubes (polystyrene, 5ml).