Koch Institute Flow Cytometry Core at MIT

Sytox Green Protocol (Yeast)
1. Spin down 1E7 yeast cells in a microfuge for one minute.
2. Resuspend pellet in one ml of 70% EtOH. Fix for at least 60 minutes at room temperature (or up to several days at 4° C).
3. Pellet cells and resuspend in 1 ml of 50mM sodium citrate, pH 7.0.
4. Sonicate (30% for 15 seconds), pellet, and resuspend in 1 ml of the same solution.
5. Add RNase A to 0.25 mg/ml. Incubate at 50° C for one hour or overnight at 37° C.
6. Pellet and wash cells. Pellet again and resuspend in 1 ml of 50 mM sodium citrate that contains 1 uM Sytox Green. (Stock is available from Molecular Probes, S-7020, and is 5 mM.) Keep at room temperature in the dark for at least one hour.
7. Analyze on flow cytometer.

Courtesy of Puck Ohi at the Kathy Gould Lab at Vanderbilt.

Propidium Iodide Protocol (Yeast)

1. Grow cells under the desired conditions to ~1 x 10^7 cells/ml (i.e. early log phase). For each analysis, 5 x 10^6 - 1 x 10^7 cells are required. All of the following steps can be performed in a single 15 ml screw cap or snap cap tube.
2. Pellet the cells in a clinical centrifuge at setting 6 for 5'. Resuspend the cells in 3 ml of dH2O.
3. At this point, one can sonicate the cells to prevent clumping observed in some yeast strains. This can lead to sharper peaks and less debris above the G2 DNA content. To sonicate the cells, I generally use the small (micro) probe and set the machine to pulse (not continuous). With the duty cycle at 35% and the output control at 2.5, I give the cells three pulses.
4. While mixing slowly on the vortexer, slowly add 7 ml of 95% EtOH to fix cells.
5. Incubate at 4° C for 2-16 hours.
6. Spin down cells as above and resuspend cells in 5 ml of 50mM sodium citrate (pH 7.4). If clumping is a severe problem, you can sonicate again at this point. (I have not found this necessary.)
7. Spin down cells as above and resuspend in 1 ml of 50mM sodium citrate containing 0.25 mg/ml boiled RNase A (final).
8. Incubate cells for one hour at 50° C.
9. Add 50ul of 20 mg/ml Proteinase K and continue the incubation for an additional hour at 50° C.
10. Add 1 ml of 50mM sodium citrate containing 16 ug/ml propidium iodide (added from a 100 X stock of 1.6 mg/ml propidium iodide prepared in advance and stored at -20° C wrapped in tin foil).
11. Incubate 30 minutes at room temperature or overnight at 4° C. At this point, the cells are ready for the FACS analysis and should be protected from light. They can be stored at 4° C for up to a week before analysis, however.
12. Analyze on flow cytometer.

Courtesy of Stephen P. Bell.

Triton X Protocol (Mammalian)

1. Spin cells for 10 minutes at 1000 RPM, and resuspend the pellet in the remaining supernatent.
2. Add stain solution at 1 ml per 2-4 x 10^6 cells.
3. Incubate for 20 minutes at 37° C.
4. Double the volume of the stain and cell mixture with the salt solution. The final concentration should be 10^6 cells/ml. 5. Store in the dark at 4° C for at least one hour before analyzing. If possible, store overnight. DO NOT FIX CV.
6. Analyze on flow cytometer.

Stain Solution (10 ml):
0.3 gm polyethylene glycol (PEG) 6000 (3% w/v)
0.5 ml PI stock final (50uG/ml)
0.5 ml RMAse stock final (180 units/ml) 0.1 ml 10% Triton X stock final concentration=0.1%. Dilute 5 ml to 50 ml with PBA (PBS + 1% BSA)
8.9 ml 4 mM citrate buffer pH 7.8 (0.588 gm diluted to 500 ml with dd water)
Adjust final pH to 7.2. NEVER CENTRIFUGE THE SOLUTION. This will increase the chance of clumping.

Salt Solution (10 ml): (Hyperosmotic--brings medium to isotonic level and they last longer)
0.3 gm PEG 6000 (3% w/v)
0.5 ml PI stock
0.1 ml 10% Triton X
9.4 ml 0.4 M NaCl (11.69 gm diluted to 500 ml with dd water)
Adjust final pH to 7.2

Alcohol Protocol (Mammalian)

1. Isolate the cells and transfer them to a 15 ml conical tube. Check for a single-cell suspension. Centrifuge at 1000g for five minutes. Remove supernatant.
2. Wash the cells twice in PBS without calcium or magnesium. At the last wash, count the total number of cells, and record the number on the tube.
3. Resuspend the pellet in approximately 500 ul of ice-cold PBS. Pipet with blue tip up and down 20 times. It is important that this be a good single-cell suspension at this point, or the cells will be fixed as clumps.
4. Add 5 ml of COLD (-20° C) ethanol, drop by drop, while vortexing to prevent clumping. (Cells can remain in fixative for up to three weeks before staining.) Fix at 4° C overnight.
5. Take 5 x 10^6 cells into a 15 ml conical tube. Centrifuge at 1000g. Remove the ethanol.
6. Vortex pellet. Wash twice in 5 ml of PBS + 1% BSA or calf serum. (Ethanol-fixed cells are difficult to pellet. Adding BSA or serum to the wash medium will overcome this.)
7. Resuspend the pelleted cells in 800ul of PBS containing 1% BSA or 1% calf serum.
8. Add 100 ul of 10x PI solution (500 ug/ml PI in 3.8 x 10^-2 M sodium citrate, pH 7.0).
9. Add 100 ul of boiled RNase A (10 mg/ml prepared in 10mM Tris-HCl, pH 7.5) and incubate at 37° C for 30 minutes. (If the samples will not be used immediately, they should be protected from light and stored at 4° C.)
10. Analyze on flow cytometer.