Aging cultures are maintained in test tubes at 30 degrees Celsius with constant agitation. Bio, A roller drum, an aliquot of cells from each aging culture is inoculated into a well of a bio screen honeycomb plate containing YEPD medium. The bio screen CMBR machine incubates and shakes the honeycomb plate for 24 hours while measuring optical density of each well.
Every 30 minutes growth curves can be generated from the data output by the bio screen, which can be used to calculate viability of each aging culture. After the 24 hour incubation cell densities in the honeycomb plate, wells can vary depending on the viability of the original aging culture. Hi, I'm Chris Murakami from the laboratory of Dr.Matt Capline and the Department of Pathology here at the University of Washington.
Today we're gonna show you procedure for quantifying geese chronological lifespan using outgrowth of age cells. We use this procedure in our lab to study how both genetic and environmental factors influence the longevity of yeast. So let's get started.
To prepare aging cultures first streak to yeast strains from frozen stocks onto YEPD auger plates incubate the cells at 30 degrees Celsius for 48 hours or until single colonies appear. Once single colonies have appeared, pick single colonies and inoculate into five milliliters of YEPD. Liquid medium.
In test tubes, grow cultures overnight at 30 degrees Celsius in a roller drum to maintain constant agitation after the overnight incubation, use 50 microliters of each culture to inoculate five milliliters of synthetic complete medium. Generally three SC cultures are prepared for each strain to provide triplicate biological replication of the lifespan analysis for each strain being examined. Maintain the cultures at 30 degrees Celsius with constant agitation on a roller drum for the entire experiment, which is generally two or more weeks after two days of culture.
In SC media, the cells should be in a stationary phase and the first age point is ready to be taken. Subsequent age points should be taken every two to three days for a minimum of two weeks. To begin this procedure, prepare the bio screen.
100 well honeycomb plates for inoculation filling each well with 145 microliters of YEPD. Be sure to leave at least one well filled with only YEPD and no cells for later data analysis. Remove the aging cultures from the incubator.
Briefly, vortex the first culture to be inoculated into the honeycomb plate. Be careful not to spill any of the culture. Flame the mouth of the test tube and remove five microliters of the culture, pipette it into the first well of the honeycomb plate.
Don't forget to flame the mouth of the test tube. After removing the five microliter aliquot, repeat this inoculation procedure for each aging culture. Be sure to note the well position corresponding to each culture identical.
Well positions should be used for each subsequent age point through the entire experiment. Return the cultures into the 30 degree Celsius incubator. When the inoculations are finished, after the cultures have been inoculated into the honeycomb plate, the plate is loaded into the bio screen CMBR machine.
To do this, expose the incubator compartment by lifting the lid. Then remove the cover to the sample tray. Insert the newly inoculated honeycomb plate into the sample tray.
Use the left slot. If you are only reading one plate, replace the cover to the sample tray and lower the lid to the incubator compartment. It is important to make sure the heat transfer fluid is above the minimum fill level.
If it is not, add more heat transfer fluid using a 1000 microliter pipette Using the bio screen software Easy experiment. Set the following parameters to obtain suitable growth curves for croce visi. For the number of samples, enter the number of wells with media or 200, set the filter at four 20 to five 80 nanometers wide.Band.
Set the temperature to 30 degrees Celsius. The experiment length should be one day, zero hours, zero seconds. The measurement interval should be 30 minutes.
And finally the shaking should be set to on continuous shaking and high click start to begin readings. Obtain the output files from the bio screen CMBR machine. The easy experiment software will output the bio screen data as a tab delimited file that is compatible with Microsoft Excel and other software.
The first column, column A shows the time the OD measurement was taken during the experiment. Subsequent columns represent each well in the bio screen, honeycomb plates inoculated with cells from the aging cultures. Delete the first OD reading from every column because this reading is noise.
Normalize the data by subtracting the optical density value of the well with YEPD alone from the optical density values in each column. This removes the background absorbance by the media plot. The outgrowth curves.
The curves will shift as a function of age, for example, by plotting the outgrowth curves from column one, well one of the honeycomb plate over the six time points, there is a distinct rightward shift of the curves over time. The length of time between the initial time point, day two and a successive time point determines viability at that particular age. Calculate the doubling time for each well based on the growth kinetics of the day two age point.
The equation used to calculate doubling time looks like this, where OD one and OD two represents successive optical density measurements and T one and T two are the time between measurements. Calculate doubling times only between OD values of 0.2 and 0.5. The average of these values is the doubling time for that.
Well, most wild type yeast strains should give a doubling time value between 85 to 90 minutes for each age point. Calculate the time shift delta T in the outgrowth curves relative to the initial age point, which is day two. An easy way to do this is to determine the difference in the length of time it took for each well to reach an OD of 0.3 between the initial age point and each subsequent age point.
The time that a particular well reached an OD of 0.3 can be calculated from the linear regression equation corresponding to the natural log optical density. Calculate the fraction surviving at each age point in order to generate a survival curve with the initial day two age point, representing 100%viability for each successive age point. Calculate the percent survival using the following equation.
Where SN is the survival percentage. Delta TN is the time shift, and Sigma N is the doubling time. Generate survival curves as desired for each.
Well by plotting the fraction of viable cells as a function of age, calculate the survival integral for each well SI is defined as the area under the survival curve and can be estimated by this formula where age N is the age point and SN is their survival value at that age point determines statistical parameters of replicate wells from the SI and survival data. Common statistical parameters of interest include mean median and variance for each set of biological replicates. A T-test or similar analysis can be used for pairwise comparison of SI for different experimental and control groups.
It may also be desirable to generate survival curves from average to biological replicates. At the completion of the experiment, you will have plotted the survival curve and performed sufficient data analysis to determine the chronological aging potential for several different strains or conditions If performed properly, the growth curves obtained from the bio screen CMBR machine should look similar to this curve. Generally, wild type cells cultured under the conditions described here will have a median chronological lifespan on the order of seven days.
Substantial variation in survival is observed in different strains and under some conditions such as growth in 0.05%Glucose media median survival can exceed 30 days. We've just shown you a high throughput method for quantitatively determining the chronological lifespan of the yeast. Croce visier.
It's important to remember that when doing this procedure, routine maintenance of the bio screen CMBR machine is important for both reproducible data and accuracy. So that's it. Good luck with your experiments.
