Koch Institute Flow Cytometry Core at MIT
SAMPLE PREPARATION GUIDELINES
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FLOW CYTOMETRY TUTORIALS
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DNA LABELING PROTOCOLS:
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.