The budding yeast Croce Visi has been used extensively to study the biology of aging. In this article, we present a general protocol for measuring the replicative lifespan of yeast mother cells. Hi, I am Kristen Steffen from the laboratory of Brian Kennedy in the Department of Biochemistry at the University of Washington.
Today we'll show you a procedure for measuring replicative lifespan of yeast mother cells. We use this procedure in our laboratory to study genes which influence the lifespan of yeast cells. So let's get started.
For the replicative lifespan experiment and the preparation of yeast cells for lifespan analysis, solid YEPD plates need to be prepared to do this. Use appropriate sterile technique to prepare these YEPD agar plates. Prepare at least two plates for every four to five strains to be analyzed in the lifespan experiment.
These plates should be prepared at least two days before the lifespan experiment and allowed to dry before beginning the lifespan assay. If the experiment will not take place within four to five days of pouring the plates, they should be wrapped in para film or plastic and be placed in a refrigerated incubator to prevent desiccation. Remove the yeast strains from frozen stocks and streak for single colonies.
Onto the YEPD agar plates. Up to six strains can easily be streaked on the same YEPD plate by partitioning the plate into equally sized pie-shaped wedges. Incubate the cells at 30 degrees Celsius for two days.
After incubation, remove the cells from the incubator and patch cells from a single colony onto a fresh YEPD plate. At this time, the strains to be analyzed should be coated to ensure the replicative lifespan experiment is performed blind. Where the dissectors do not know the identity of individual strains.Chains.
Next, incubate the patched coated cells at 30 degrees Celsius until the following evening. Remove the cells and lightly patch cells from each coated strain to fresh YEPD plates. These will serve as the experimental plates used for the replicative lifespan.
Analysis cells should be patched along a vertical line on the left side of the YEPD plate. Do not transfer too many cells onto a single fresh YEPD plate. Since this will cause thick growth of the cells during the overnight incubation and affect their replicative lifespan, approximately three to five patches per plate is optimal.
Assuming replicative lifespan analysis will be performed on 20 cells from each patch, Now the lifespan experiment is all set up and all they have to do is incubate overnight on the bench top. And then tomorrow we can start the micro dissection To position the yeast cells on the plate and obtain virgin daughter cells. For replicative lifespan analysis.
Use a microscope equipped with a micro dissection apparatus suitable for yeast. Transfer approximately 50 cells from the first patch strain to a position on the plate distal to the patched cells. If your microscope stage is graded, these gradations can be used to ensure the cells will be easy to find during subsequent iterations.
Sometimes cells can become stuck in the needle, which can be a problem if they come out at the wrong time. To make sure the needle is clean and free of cells, touch it to the agar surface repeatedly. Then create a hole in the agar by forcing the needle through the agar surface.
This hole will serve as a marker to orient you on the plate during subsequent iterations of dissection and dotter cell removal. Be careful not to get yeast cells in the hole, or they will grow and form a colony next directly above the hole. Vertically aligned individual yeast cells into 20 evenly spaced positions with one to three needle diameters between each cell position.
For every few cells, place two cells next to each other in the same position in the line rather than one. This allows for redundancy during virgin daughter cell selection. If one of the transferred cells fails to divide between the 10th and 11th positions in the line, create a horizontal series of seven to eight holes in the agar.
This will serve as a marker to orient you on the plate during subsequent dissections and daughter cell removals. Again, be careful not to get yeast cells in the holes or they will grow and form a colony. Repeat this procedure for each patch on the plate, continuing to array the cells vertically along the same axis.
It is important to remember that when creating the hole in the AER to have the number of holes created correspond to the patch number so that there is one hole for the first patch, and two holes for the second. Once the cells have been arrayed for each patch, paraform the plate and incubate at 30 degrees Celsius for approximately two hours. Remember, from this point on, all plates should be para filmed except when undergoing dissection.
In order to prevent desiccation, repeat this procedure for each plate. In the experiment. For the lifespan analysis, it is important to be sure that each cell starts as a virgin daughter cell.
To do this first, remove the plates from the incubator and verify that most of the array cells have undergone a cell Division. So what we have here are cells that have been aligned previously, and you can see that after two hours in the incubator, they've begun to divide. And now we have a mother cell, the larger one attached to its body, the virgin daughter cell, and that virgin daughter cell is a cell that will be used to begin the lifespan.
If additional time is needed to allow cell divisions to be completed, return the plate to the incubator. Beginning with the first array cell. Use the needle to detach daughter cells from the mother cell by gently placing the needle on top of the attached cells.
It is sometimes helpful to tap the side of the microscope base while the needle is resting on the cells. Next place the detached daughter cell in the vertical line of cells replacing the mother cell and any additional daughter cells and moving them temporarily aside. Repeat these steps for each of the array cells.
If a cell fails to divide, take a daughter cell from one of the redundant cells positioned next to the test cell. When finished, you should have 20 daughter cells for each patch aligned vertically on the lifespan plate. Collect all of the mother cells and extra daughter cells into a pile and transfer them back to an area near the patches called the graveyard.
It is important to keep unwanted cells far from the cells undergoing lifespan analysis to prevent formation of micro colonies and depletion of local nutrients. Once the cells have been separated, para fromm the plate and incubate at 30 degrees Celsius for approximately two hours. Repeat these steps for each plate in the experiment.
To measure the replicative capacity of individual cells, you'll need to prepare lifespan data sheets. A lifespan data sheet can be a simple grid where each row corresponds to an individual mother cell and each column corresponds to an age point. Be sure to label each data sheet according to the number of strains to be analyzed.
To begin, first, remove the plates from the incubator and verify that most of the array cells have undergone at least one cell division. If additional time is needed to allow cell division to be completed, return the plate to the incubator. Start with the first array cell on the first plate, and visually observe the mother and daughter, cell or cells to determine the number of cell divisions that have occurred.
It is generally easy to determine how many daughter cells a mother has produced based on characteristic patterns of cell arrangements. This one's really simple to see how many cells it produced because there are two there and they're the same size and they're both smaller than the mother. Record the number of daughter cells produced by the mother cell in the appropriate box on the lifespan score sheet.
Next, use the needle to detach daughter cells from the mother cell as described previously by gently placing the needle on top of the attached cells and tapping the side of the microscope base while the needle is resting on the cells. Keep the mother cell in its position in the vertical line and move the daughter cell or cells temporarily aside. Repeat these steps for each of the array cells.
Finally collect all of the discarded daughter cells and move them to the graveyard located near the original patches. Para film the plate and incubate at 30 degrees Celsius for two to three hours. Repeat the discarding of daughter cells and incubation.
Step for each plate in the experiment. Repeat this entire procedure until all mother cells have stopped dividing. Keep in mind that as the mother cells age, the cell cycle progression becomes slower and it will be desirable to incubate the plate for longer periods of time between age points afterwards, the plates can be placed at four degrees Celsius overnight.
The raw data produced by a replicative lifespan experiment is a list of numbers corresponding to daughter cells produced by each mother cell at each age point. As seen here. By summing each row of the score sheet, the replicative lifespan for each mother cell is obtained.
Data from other cells from the same experimental group can be pooled to generate a survival curve and to perform statistical calculations. The curve should be roughly consistent with Gomperts Mac am kinetics, showing a sigmoidal shape with low early mortality, followed by a relatively rapid decline in survival and a flattening out of the curve at later ages. We've just shown you how to perform a yeast replicative lifespan experiment.
When doing this procedure, it's important to remember that the cells will continue to divide whenever they aren't in the cold. So remember to put the plates at four degrees overnight so you don't end up with too many daughter cells to count in the morning. So that's it.
Thanks for watching and good luck with your experiments.
