Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

The overall goal of the following experiment is to determine whether changes in mutation frequencies with increased replicative. Age of yeast cells are due simply to additional rounds of cell division or to age specific changes in rates of accumulating mutations. This is achieved by first measuring mutation frequencies and rates in young cells by spreading cells onto selective and non-selective media to calculate a predicted mutation frequency for cells of increasing ages due to mutation accumulation with each round of cell division.

Next, a yeast cell population is labeled with biotin grown in liquid culture and the original labeled cells are recovered by magnetic sorting to obtain aged mother cells that have undergone several cell divisions. Then aged mother cells are either regrown to increase their age and then sorted again or stained for butt scars, and then spread onto selective and non-selective media in order to determine their average replicative age and mutation frequency results are obtained that show whether there are any age specific increases or decreases in accumulating mutations based on a comparison of the experimentally observed mutation frequency to the predicted mutation frequency for cells of the observed average age. This method can help answer key questions in the field of aging, such as do changes in the rates of accumulating genetic damage contribute to normal aging, or are the lifespan altering effects of various genetic and environmental factors, partly due to changes in mutation rates.

Demonstrating the procedure will be Melissa Patterson, a graduate student in my laboratory, To establish a baseline mutation rate per cell generation using fluctuation tests for cells grown under standard culture conditions. For each replicate culture dilute cells one to 2000 and spread one to four microliters onto non-selective medium to determine colony forming units per milliliter. Pellet the remaining cells at 2, 400 Gs for one minute in a micro centrifuge and use 100 microliters of water to resuspend them before spreading on selective medium to identify mutants.

After incubating the plates at 30 degrees Celsius for three days, count the colonies determine the initial mutation rate and frequency according to the accompanying text protocol to label charmy CCI with biotin Begin by streaking the culture from a glycerol stock onto YPD medium and incubate at 30 degrees Celsius for one to three days. Using a sterile pipette tip transfer cells from the plate to at least one milliliter of water. Keeping in mind that one to one and a half centimeters of a thickly grown portion of the streak will give approximately 10 to the eight cells.

Use a hemo cytometer to determine the exact cell density for each strain or treatment condition. Use a five millimolar biotin reagent stock to label 10 to the eight cells per collection point according to the manufacturer's standard procedure. Perform all centrifugation steps for five minutes at four degrees Celsius after the final wash.

Use water to suspend the labeled cells to a final concentration of 10 to the eight cells per milliliter to check cell viability to dilute 10 microliters of cells with 23 microliters of water and 67 microliters of 0.4%Triam blue in one XPBS incubate the cells for 40 minutes at room temperature before using a hemo cytometer to score live or unstained and dead or stained cells. After measuring baseline values for cell age and mutation frequency, according to the text protocol, transfer the biotin labeled cells to 20 milliliters of YPD medium. Pretend to the eight cells.

Grow the cultures for 16 to 18 hours at 20 degrees Celsius to about 10 to the eight cells per milliliter. Do not allow the cells to reach stationary phase and if necessary, keep the cells at four degrees Celsius for several hours to optimize timing of the growth period and collection. After determining the cell density and pelleting the cells at four degrees Celsius, wash cells by adding 30 milliliters of bead labeling, buffer and vortexing, then spin the suspension and pour off the supernatant.

After a second wash, we suspend the cells in 50 microliters of bead labeling buffer. Pretend to the eight total cells by vortexing. Add two microliters of magnetic beads, pretend to the eight total cells and after vortexing briefly incubate on ice for 10 minutes inverting periodically.

Next, use 30 milliliters of bead labeling buffer to wash the cells three times. Then add 0.5 milliliters of bead labeling buffer. Pretend to the eight total cells and briefly vortex the pellet on a medium setting just until the cells are resuspended.

Pour the suspension through a 40 micron cell strainer into a 50 milliliter tube to remove any remaining cell clumps. If needed, leave cells on ice for one to two hours prior to loading on the column. Follow the manufacturer's recommended protocol for the magnetic columns using up to two times 10 to the nine total cells per.

Take care to remove any airable when loading the columns and remove and replace the column on the separator between washes to prevent cells from becoming trapped between the beads. When using multi column separators for processing multiple columns of the same sample, elute them all into the same collection tube to reduce handling time and decrease loss of cells if desired. Save the flow through from the binding and washing steps to analyze young cells produced by the mother cells after concentrating the cells by centrifugation at 3000 Gs for five minutes at four degrees Celsius, aspirate all but one milliliter of buffer.

Pretend to the eight original biotin labeled cells. Then vortex briefly to suspend the cells in the remaining buffer. Use triam blue to stain an aliquot of diluted cells and use a hemo cytometer to determine cell density and viability.

Then calculate the total number of cells recovered. If the number of cells is substantially higher than expected, pass the elution sample through another column to try to remove contaminating young cells. If the number of cells is substantially lower than expected, pass the saved flow through.

Sample through another column to try to improve the yield of mother cells if desired. Regrow cells to increase the replicative age and follow the bead labeling and sorting without biotin labeling. To determine replicative age transfer 25 microliters of recovered cells into a 1.5 milliliter centrifuge tube and use one milliliter of one XPBS to wash the cells twice to label bud scars on the surface of the cells.

Use 180 microliters of one XPBS and 20 microliters of fluorescently conjugated WGA to resuspend the cells cover the 1.5 milliliter centrifuge tube with foil and incubate with gentle rocking at 30 degrees Celsius for 30 minutes after the incubation. Use one XPBS to wash the cells three times. Then for fluorescent microscopy, use an Antifa reagent to mount the cells on slides to avoid cell movement During imaging.

Fully cure the slides in the dark for up to a few days. Count bud scars on at least 50 cells per sample to obtain a representative measure of cell age. Alternatively, after suspending the cells in one XPBS perform flow cytometry using the settings outlined in the text protocol graph bud scars on young cells and age cells counted by microscopy on the Y axis against the normalized geometric mean of the WGA conjugate fluorescence from the same cell populations on the x axis.

Finally, obtain the equation of a linear trend line for this relationship For subsequent substitute, the normalized geometric mean of WGA conjugate fluorescence intensity of a cell population for X in the equation and solve for Y to determine the average cell age of a sample. This graph illustrates that the mutation rate in the can one gene was similar before and after biotin labeling In independent young yeast populations can one mutation frequencies were also similar in young cells before and after biotin labeling when grown at either 20 degrees Celsius or 30 degrees Celsius. The graph shown here demonstrates what may be observed when counting recovered cells after biotin labeling and sorting of untreated cells or cells treated with one millimolar hydrogen peroxide after the first sort.

The number of cells may appear higher than expected after the first sort, and a small progressive loss of cells is expected with continued sorting. Replicative age was determined by manual counting of fluorescently labeled, but scars on cells as demonstrated here as seen in this figure. The fluorescent but scar signal from flow cytometry was compared to manual counts to obtain a linear relationship that allows replicative age of mother and control cell populations to be determined by flow cytometry.

These graphs show examples of similar or elevated observed mutation frequencies of aged mother cells compared to the predicted frequencies calculated from the average age of the mother cells and the mutation frequencies and rates obtained for young cell populations. Depending on genomic location of the can one gene Following this procedure. Other methods can be used like staining for reactive oxygen species or ular morphology to answer additional questions like what changes in cell physiology are associated with age-related changes in mutation accumulation.