This protocol allows a precise determination of S-phase duration in synchronized budding yeast cells. It is a quick, easy, and reproducible method. In addition, it is sensitive enough to detect mild synthesis defects undetected by classical protocols.
This method will be useful for many researchers working on cell cycle regulation in budding yeast. Demonstrating this protocol will be Nicolas Talarek, a senior researcher, and Juan De Dios Barba Tena, a PhD student in the lab. Inoculate Saccharomyces cerevisiae cells in 10 milliliters of SC medium at a low cell concentration for an overnight culture at 30 degree Celsius with orbital agitation at 130 rotations per minute.
The following day, dilute the cells in 20 milliliters of fresh SC medium at a final concentration of two to three times 10 to the six cells per milliliter. At 40 microliters of one milligram per milliliter alpha factor diluted in water. Then culture the cells at 30 degree Celsius, with orbital agitation at 130 rotations per minute for one hour.
Repeat this step once. Visualize the cells under a light microscope to monitor G1 arrest. Proceed if more than 90%of the cells display a shmoo, and the others are rounded unbutted cells.
Centrifuge the samples for three minutes at 1, 500 G.Then discard the supernatant with the vacuum pipette and resuspend the cells in 20 milliliters of SC medium. Repeat this step once. Collect one milliliter of cells twice every five minutes and proceed to EDU labeling.
Add 400 microliters of the alpha factor 30 minutes after the release. Transfer one milliliter of the cell culture to a two milliliter microfuge tube containing one microliter of 10 millimolar EDU, mixed well by inversion. To discriminate the EDU positive cells from the EDU negative cells on a Pi EDU bivariant facts, transfer another one milliliter of cell culture to a two milliliter microfuge tube containing one microliter of DMSO.
Incubate for three to five minutes at 30 degree Celsius under agitation in a shaking water bath. Stop the reaction by adding 100 microliters of 100%ethanol. Pellet the cells for two minutes at 10, 000 G in a microfuge.
Remove the supernatant using a vacuum pipette. Resuspend the cell pellet in 500 microliters of 70%ethanol, and mix well by vortexing. Leave the samples at room temperature for at least one hour at 20 tilts per minute on a variable speed rocker to permeablizie the cells.
Pellet the cells for two minutes at 10, 000 G in a microcentrifuge and discard the supernatant with a vacuum pipette. Wash the cells twice with 500 microliters of 10%ethanol and PBS. Pellet the cells for two minutes at 10, 000 G in a microfuge and discard the supernatant with a vacuum pipette.
Resuspend the pellet in 200 microliters of PBS containing RNase A and Proteinase K.Incubate from one to two hours at 50 degree Celsius with occasional shaking, or overnight at 37 degree Celsius. Pellet the cells for two minutes at 10, 000 G in a micro centrifuge and discard the supernatant with a vacuum pipette. Wash the cells with 500 microliters of PBS.
Then pellet the cells and discard the supernatant as demonstrated earlier. Resuspend the cell pellet in 200 microliters of PBS with 1%BSA. Incubate for 30 minutes at room temperature.
Then pellet the cells, discard the supernatant as demonstrated earlier, and resuspend the pellet in 300 microliters of PBS containing 1%BSA. Distribute the cells into two, 1.5 milliliter microcentrifuge tubes. The first one should consist of 200 microliters of cell culture for the Click reaction, and the second tube should contain 100 microliters of cell culture for the sytox green staining.
Then, pellet the cells and discard the supernatant as demonstrated earlier. For sytox green staining, resuspend the cell pellet in 100 microliters of PBS. Then depending on the cell concentration, transfer 10 to 30 microliters to a flow cytometer tube containing 300 microliters of sytox green mixture.
Sonicate the samples twice for two seconds at an amplitude of 40 to 50%Leave in the dark until processing the samples on a flow cytometer. For the Click reaction, resuspend the cell pellet with 40 microliters of freshly prepared Azide dye buffer. Incubate at room temperature in the dark for 60 minutes.
Pellet the cells and discard the supernatant as demonstrated earlier. Then wash the cells three times with 300 microliters of 10%ethanol in PBS. Next resuspend the cells in 100 microliters of 50 micrograms per milliliter propidium iodide in PBS and leave for 10 minutes in the dark.
Depending on the cell concentration, transfer 10 to 30 microliters of the cell suspension to a flow cytometer tube containing 300 microliters of 50 millimolar Tris HCl, pH 7.5. Sonicate twice for two seconds at an amplitude of 40 to 50. Leave the samples in the dark until processing them on a cytometer.
Read the sytox green samples using an excitation blue laser at 488 nanometers, and a 530 by 30 band pass filter. Read the Bavaria Pi EDU samples on a dot plot using an excitation blue laser at 488 nanometers and a 615 by 20 band pass filter for the Pi X-axis, and a red excitation laser at 640 nanometers, a 660 by 20 band pass filter for the Y-axis. Three populations of cells were observed in the bivariant EDU Pi cytometer analysis.
At 25 degree Celsius, approximately 27%of the cell population was in the G1 phase, 29%was in the S phase, and about 44%was in G2 plus M phase. In synchronized cells, the duration of the S phase may be about 25 minutes based on the time when the DNA content changed from one C to two C on the sytox green flow cytometer profile. EDU pulse labeling accurately determined the S phase duration.
15 minutes after the release, a fraction of EDU positive cells was detected, indicating the start of the S phase. The progression through the S phase was seen by the cell cloud moving upward and rightward in the Bavaria Pi EDU graph. Finally, at 35 minutes from the release, a fraction of cells was EDU negative, but with twice the amount of DNA indicating that the S phase ended and the cells were in the G2 plus M phase.
Despite the high synchrony observed, some cells finished the S phase 60 minutes after the release and the S phase was complete for the whole population approximately 65 minutes after the release. It is crucial to have a perfect synchronization and to prevent cells entering the second S phase. Cells can be imaged with a microscope where nuclear DNA replication foresight can be monitored and quantified.
This technique will help to identify overlook mutants that have mild S phase defects, as well as cell cycle duration defects.
