Flow Cytometry Immune Monitoring Shared Resource


Yeast Cell Cycle with Sytox Green

  1. Grow cells under the desired conditions to 0.5-1x107 cells/mL. Collect up to 1-2x107 cells per sample. All the following steps can be performed in a single 1.7mL microfuge tube per sample.
  2. Pellet the cells (a few seconds in a microfuge). Resuspend the cells in 300μl H2O. Be sure the pellets have been dispersed before the next step.
  3. Add 0.7mL 100% ethanol and vortex; incubate at 4°C for at least 30 minutes.
  4. Pellet the cells as before and resuspend in 1mL of 50mM 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 1mL Na-citrate containing 0.25mg/mL RNase A. Incubate at 50-55°C for 1 hour.
  7. Add 25μL of 20mg/mL Proteinase K and continue the incubation for an additional hour.
  8. Pellet the cells again and resuspend the pellets in 500μl of 50mM 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 (= 1μM) in 50mM Na-citrate. The stock solution provided by the vendor is 5mM = 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 50μL of the cell sample (or enough to get ~1-2x106cells; 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 (10mg/mL)

  • 0.2g Ribonuclease A (Sigma #R-5503)
  • 200µL 1M Tris-HCl, pH 7.5
  • 60µL 5M NaCl
  • Bring volume up to 20mL with H2O
  • Boil 15 minutes.
  • Let cool slowly to room temperature.
  • Aliquot and store at -20°C.

Proteinase K Stock Solution (20mg/mL)

  • Stock solution is 20mg/mL in
  • 10mM Tris pH 8.0
  • 1mM CaCl2
  • 50% glycerol
  • To make 5mL of stock solution, dissolve 100mg Proteinase K in:
  • 3.125mL 80% glycerol*
  • 50μl 1 M Tris pH 8.0
  • 50μl 100mM CaCl2
  • 1.775mL H2O
  • = 5mL total; dispense in 1mL portions and store at –20°C.
  • *autoclaved stock; easier to deal with than 100% glycerol that’s not been sterilized