1. Grow cells under the desired conditions to 0.5-1 x 107 cells/ml. Collect up to 1-2 x 107 cells per sample. All the following steps can be performed in a single 1.7 ml microfuge tube per sample.
2. Pellet the cells (a few seconds in a microfuge). Resuspend the cells in 300 μl H2O. Be sure the pellets have been dispersed before the next step.
3. Add 0.7 ml 100% ethanol and vortex; incubate at 4°C for at least 30 minutes.
4. Pellet the cells as before and resuspend in 1 ml of 50 mM sodium citrate, pH 7.4.
5. Sonicate the cells (10 pulses of 0.9 seconds each, setting of –32 to –40).
6. Pellet the cells again and resuspend them in 1 ml Na-citrate containing 0.25 mg/ml RNase A. Incubate at 50-55°C for 1 hour.
7. Add 25 μl of 20 mg/ml Proteinase K and continue the incubation for an additional hour.
8. Pellet the cells again and resuspend the pellets in 500 μl of 50 mM Na-citrate. [But go instead to “Alternative” in Step 10 if your cell concentration is not very high.]
9. Prepare a batch of 1x Sytox Green (= 1 μM) in 50 mM Na-citrate. The stock solution provided by the vendor is 5 mM = 5000x, in DMSO. Note: the manufacturer recommends wearing two pairs of gloves when handling the stock solution. The Sytox stock solution takes a while to thaw, so plan ahead.
10. Pipette out 1 ml aliquots of the 1x Sytox solution into FACS analysis tubes (Falcon 2054). Add 50 μl of the cell sample (or enough to get ~1-2 x 106 cells; the goal is to have 100-200 events per second within the gated region when the samples are being read). Incubate 1 hour to overnight at 4°C; stained samples can be stored at 4 °C for several weeks. [Alternative: If your sample is low-concentration, you can simply add 1 ml of 2x Sytox to the cell sample immediately after step 7, and you’ll be good to go.]
11. Use FL1 on the FACSCAN instrument. Sytox Green fluoresces more strongly than propidium iodide, so the voltage/gain won’t have to be set as high as with propidium iodide.
Sytox Green: Invitrogen, catalog # S7020
RNase A stock solution (10 mg/ml)
0.2 g Ribonuclease A (Sigma #R-5503)
200 uL 1 M Tris-HCl, pH 7.5
60 uL 5M NaCl
Bring volume up to 20 ml with H2O
Boil 15 minutes.
Let cool slowly to room temperature.
Aliquot and store at -20°C.
Proteinase K stock solution (20 mg/ml)
Stock solution is 20 mg/ml in
10 mM Tris pH 8.0
1 mM CaCl2
To make 5 ml of stock solution, dissolve 100 mg Proteinase K in:
3.125 ml 80% glycerol*
50 μl 1 M Tris pH 8.0
50 μl 100 mM CaCl2
1.775 ml H2O
= 5 ml total; dispense in 1 ml portions and store at –20°C.
*autoclaved stock; easier to deal with than 100% glycerol that’s not been sterilized