The overall goal of the following experiment is to quantify mitogen activated protein expression at the single cell level. To achieve this, a yeast strain bearing the fluorescent expression reporter quadruple Venus controlled by the STL one promoter and specific to the activation of the hog one map kinase was generated. Fluorescent protein expression can be quantified either by a live cell microscopy assay in which the cells are stressed directly on the microscope to facilitate real-time apparition of the fluorescence or by flow cytometry, in which case the cells are stressed in a micro tube and the protein synthesis is blocked at a given time.
By the addition of cyclo heide, only a few cells at a time can be analyzed with live cell microscopy, but the fate of the individual cells can be directly observed and correlated with other cellular property flow cytometry on the other hand, also allows assessment of mitogen activated protein expression on large numbers of cells at once. This method can help answer key questions in the signaling field, such as which proteins are involved in the regulation of gene expression. Though this method can provide insight into the yeast pathway, it can also be applied to other signaling cascade depending of the availability of the proper expression.Reporter.
To prepare the cells for live cell microscopy, begin by inoculating five milliliters of synthetic medium with yeast and then grow the cells at 30 degrees Celsius overnight. The next morning, measure the OD 600 of the overnight culture and then dilute the overnight culture in five milliliters of synthetic medium to an OD 600 of 0.05. Grow the diluted culture at 30 degrees Celsius for at least four more hours.
Next, filter about 200 microliters of freshly prepared conna valent a solution into the well slide. After 30 minutes, remove the con a solution and add 150 microliters of water to the well. Now remove the water and let the well dry while the cells are prepared.
Measure the OD 600 of the cell culture again, and then dilute the cells to obtain 300 microliters of cell culture at an OD 600 of 0.02 and a 1.5 milliliter micro tube. Sonicate the cells in a water bath for 45 seconds. Briefly vortex the micro tubes and then sonicate and vortex the cells again.
Now add 200 microliters of the cells to the well slide, and then after letting the cell settle to the bottom of the well for about 30 minutes, place the slide onto the microscope. Next, select the illumination settings for their fluorescent channel to be recorded and for two bright field images, adjusting one of the bright field settings approximately three microns below the focal plane to allow for proper cell segmentation. Then set the time intervals for the time lapse measurements and select the fields of view for imaging.
Now start the acquisition for a few frames and then pause the acquisition to add 100 microliters of freshly prepared 0.6 molar sodium chloride medium, and resume the imaging to prepare the cells for measurement by flow cytometry. Begin by inoculating the yeast in five milliliters of synthetic medium and grow the cells overnight at 30 degrees Celsius. The next morning, measure the OD 600 of the overnight culture and then dilute the cell suspension in five milliliters of synthetic medium to an OD 600 of 0.1.
Grow the culture for at least four hours at 30 degrees Celsius to reach an OD 600 of 0.2 to 0.4. Then add 100 microliters of freshly prepared 0.6 molar sodium chloride to a 1.5 milliliter micro tube for each time point to be measured at time zero. Add 200 microliters of cells to each micro tube and incubate the cultures with shaking at 30 degrees Celsius.
Then at each of the experimental time points, add 30 microliters of freshly prepared cyclo heide to one of the micro tubes. While the micro tubes are incubating, add 400 microliters of PBS to one corresponding fax tube for each micro tube. After the incubation, briefly vortex the micro tubes and then sonicate the cells and add water bath for one minute as just demonstrated.
Then add 100 microliters of cell culture from each micro tube to its corresponding fax tube at the flow of cytometer. Load the proper excitation and detection settings and test the non expressing and fully expressing samples to verify that they fall in the detector sensitivity range. Finally, measure 10, 000 cells for each sample in these images, yeast cells bearing the expression reporter quadruple venous protein under the control of the TL one promoter and attached to the bottom of a well slide were stimulated by direct edition of stress medium.
As just demonstrated, the cells were monitored for about two hours at 10 minute intervals, and images were acquired both before and after edition of the stimulus. Note, the fluorescent expression of the report during the activated cells. To extract the quantitative information contained in these images, a complex image analysis process must be performed here.
The result obtained with the yeast quant analysis platform are shown. Each red trace represents the temporal evolution of the average fluorescence intensity of a single cell. The blue line illustrates the median of more than 500 cells that were segmented and tracked throughout 20 frames of five time lapse movies acquired from five different fields of view from the same well the light blue area represent the 25th and 75th percentile of all the intensities measured at a given time point to study the dynamics of the expression of the same reporter by flow cytometry Cycloheximide was added to the yeast cell cultures at different time points at five to 10 minute intervals.
The drug blocks the synthesis of new proteins, but the maturation of the fluorescent protein already expressed is not perturbed. Therefore, the protein expression visualized by the displacement of the histogram to the right can be quantified at the time of Cycl H edition circumventing the problems associated with fluorescent protein maturation. In this graph, the dynamics of the expression reporter as measured by microscopy or flow cytometry are compared while the fluorescent signal rises 30 to 40 minutes after edition of the stress medium as measured by the microscopy, the flow cytometry measurements clearly indicate that the proteins are already produced between 10 to 15 minutes after the stimulus demonstrating the slow maturation of the fluorescent proteins.
Both techniques allow access to single cell information for these simple experiments, the variability between three biological replicates is small compared to the large variability observed in the single cell responses by microscopy or flow cytometry. Following these procedures, other methods like those response measurements can be performed in order to answer additional questions such as, which is the threshold for gene expression. After watching this video, you should have a good understanding of how to measure protein expression at single cell level with microscope of flow cytometry.
