The aim of this procedure is to monitor how proteolysis of cycling B governs anaphase onset and mitotic exit. This is accomplished by first seeding cycling B snap reporter cells on microscopy slides. The second step is to label the chimeric Cyclin B snap reporter molecule with the fluorescent substrate TMR star.
Next, the image series are acquired at a microscopy station. The final step is to analyze cells and calculate TMR star fluorescence intensity over time, which reflects cycling B proteolysis. Ultimately, immunofluorescence microscopy of living reporter cells reveals the kinetics of cycling B proteolysis taking place during mitosis.
The main advantage of this technique over existing methods like monitoring cyclone BGFP, is that cyclone B SNAP allows monitoring of cyclone B degradation rather than whole protein expression. The snap reporter cells for this experiment are initially allowed to grow asynchronously in log phase for at least 48 hours. To begin, the procedure for seeding trypsin is the sub confluence snap reporter cells for seeding of cells onto eight well microscope chambers at a constant distribution across the entire surface of the chamber centrifuge.
10, 000 cells and Resus spend in 350 microliters, a phenol red free normal growth medium. Transfer the cell suspension to the microscope chamber for seeding of cells onto eight well microscope chambers with a maximum cell density in the center of the chamber. First load the chamber with 300 microliters of pheno red free normal growth medium.
Next, add 5, 000 cells carefully to the center of the microscope chamber for seeding of cells onto 96. Well special optics plates at a constant distribution across the entire surface of the well centrifuge, 5, 000 cells and resuspend in 300 microliters of phenol red free normal growth medium depending on the total number of cell containing wells required. Adjust the cell number and the total volume of the suspension medium.
Then transfer the cell suspension to a 96 well plate for seeding of cells onto 96. Well special optics plates with maximum cell density in the center of the well. Carefully add 1, 500 cells in 15 microliters of phenol red free normal growth medium in a small drop to the center of each well.
This will restrict cell growth to the center of the well. Allow all seeded cells to grow for at least 18 hours under standard cell culture conditions 30 minutes prior to the beginning of the staining procedure. Warmup quats of phenol red free normal growth medium to 37 degrees Celsius.
The snap substrate in this demonstration is TMR star for easy handling of the TMR star dissolved TMR star in DMSO to obtain a stock solution with a concentration of 400 micromolar dilute 0.5 microliters of the TMR star stock solution. In 200 microliters of warm phenol red free normal growth medium to obtain a final labeling concentration of one micromolar. Next, remove normal growth medium from the asynchronously growing cells and replace with labeling medium incubate cells in labeling medium for 25 minutes under standard culture conditions.
After 25 minutes, remove labeling medium and wash cells four times with warm phenol red free normal growth medium. After the final wash, incubate cells in 300 microliters of warm phenol red free normal growth medium for 30 minutes prior to transporting to the microscope. Replace the medium with fresh, warm phenol red free normal growth medium to remove residual unbound snaps substrate transport cells to the microscope in a styrofoam box on a prewarm 37 degree Celsius heat block.
To minimize temperature variation two hours prior to the measurement of fluorescence intensity. Adjust the air temperature of the climate chamber to 37 degrees Celsius in dry mode in order to bring the microscope and all its components to the desired temperature. To avoid condensation and subsequent damage to the microscope, it is important to preheat before setting the humidity.
When the entire microscope system has reached 37 degrees Celsius, adjust air humidity to 60%and carbon dioxide to 5%Start the scan or acquisition software and define standard settings. Define the positions of the wells to be analyzed. Define the delta T or acquisition cycle time and the absolute number of acquisition cycles.
In this demonstration. Hardware autofocus is preselected for analysis of a higher number of wells. Start the acquisition and supervise for the first two acquisition cycles.
The microscope will focus on the histone H two GFP signal with subsequent acquisition of a first image in that channel before the filter is changed and the corresponding TMR star image is acquired. This is them repeated for all positions within a well and for each of the wells to be examined before repeating the next cycle.Again. To analyze proteolytic profiles, start the scan R analysis software.
Analyze the images with the cell nuclei as visualized by histone H two GFP, defined as the main object using a threshold based on signal intensity and a watershed algorithm to assist in separating neighboring cells. A sub object consisting of nucleus with cytoplasm should be created for TMR star analysis. Important properties of the main object are X and Y positions, time and maximum, and mean intensities of GFP for the main object and the total intensity divided by area.
For the TMR star sub object, change to trace mode to visualize the sub object mean TMR star fluorescence intensity over time or cell traces assigned to the analyzed main objects. Looking at larger numbers of cells allows a first representative and objective view, but the number of cells to be examined can be narrowed down by gating on those measurements lasting at least 140 cycles and containing a large maximum histone H two GFP intensity. Select a cell trace of interest to visualize histone H two GFP and TMR star fluorescent simultaneously at the single cell level.
Using the right mouse, click on a cell of interest and generate an exportable picture. Gallery for illustration of histone H two GFP and cycling B snap TMR star for every single time. Point change to the population mode of the scanner analysis software and gate the region where the cell of interest is represented on the dot blot.
Apply a new dot plot window to the gated region and visualize mean TMR star fluorescence intensity over time. Export data to Microsoft Excel for further calculations. The figures here depict Cyclin B kinetics represented by a TMR star fluorescence intensity curve of a cell that proceeds through a regular mitosis without signs of chromosomal misalignment upon compaction of the cytoplasm following nuclear envelope breakdown or NEBD as indicated by the red triangle TMR staph fluorescence intensity shows an abrupt increase until the isomorphic window is reached.
When the cell enters prometaphase fluorescence intensity remains at a stable level as long as the cell proceeds through prophase and metaphase, and then starts to drop rapidly. Once all of the chromosomes have established a stable metaphase plate, this drops precedes chromosome separation. During anaphase indicated by the blue dot on the curve in late mitosis, the chromatin starts to decon condense and the cell adopts into phase morphology.
While the fluorescence intensity curve approaches the plateau, which is lower than the plateau before mitosis After its development. This technique paved the way for researchers in the mitosis field to explore the tight balance between cycling, be degradation and synthesis that regulates mitotic progression.
