An Assay for Cell Cycle Analysis of Antigen-Specific CD8+ T Cells
Transcripción
Prepare fresh 1X permeabilization wash buffer by diluting 10X permeabilization buffer with distilled water, and filter it through a 0.45-micrometer filter to eliminate aggregates. Dilute monoclonal antibody Ki67-FITC in 1X permeabilization wash buffer as determined with a titration experiment for a final volume of 100 microliters per sample.
Add 3 milliliters of the 1X permeabilization wash buffer to each tube with cells and centrifuge at 400 g for 5 minutes at room temperature. Discard the supernatant. Again, add the 1X permeabilization wash buffer and centrifuge to pellet the cells, then, discard the supernatant.
Add 100 microliters of diluted monoclonal antibody Ki67-FITC to the pellet, then, vortex and incubate for 30 minutes in the dark. After the incubation, wash the cells twice with 4 milliliters of 1X permeabilization buffer, centrifuging after each wash, and discard the supernatant.
Add 350 microliters of PBS to the cell pellet for samples to be acquired directly at the flow cytometer, and 250 microliters of PBS to the cell pellet for samples to be incubated with Hoechst for DNA staining. Prepare Hoechst and PBS solution and add it to each sample, so that the final concentration is 2 micrograms per milliliter. Vortex and incubate the samples for 15 minutes in the dark. Then, centrifuge the samples for 5 minutes and add 350 microliters of PBS to the cell pellet.
Open the software and create different groups corresponding to the different organs to be analyzed by clicking Create Group in the workspace ribbon section. Open the Modify Group window by double-clicking on the group name and synchronize the newly created groups by inserting a check mark on the function Synchronized.
Drag each FCS file to its corresponding group, then, create a gating strategy starting with a through LN group. Open the graph window by double-clicking on the fully stained sample to display the ungated events acquired for this sample in a dot plot. Note that the x and y-axis are labeled as in the FCS files, as indicated in Table 2 of the flow cytometer settings file of this manuscript. Check that a sufficient number of events is displayed for appropriate visualization.
If necessary, open Preferences by clicking on the heart icon in the workspace ribbon, select Graphs, then Dot Plot as Graph Type, and check that the number of dots drawn in the corresponding box is correct or change it. Display the ungated events in the graph window in a dot plot with DNA-A on the x-axis and DNA-W on the y-axis. Use linear scale for both axes. If necessary, change scale from logarithmic to linear by clicking on the box indicated with a T close to each axis in the graph window.
Select a single cell population by clicking on Rectangle in the Gating Tools section, then double-click in the center of the rectangular gate to display single cells in a dot plot with FSC-A parameter on the x-axis and dead cell dye on the y-axis.
Click on the Polygon to select the live cell population, which are negative for dead cell dye. Double-click in the center of the polygon gate to display the cells in a dot plot with the FSC-A parameter on the x-axis and the SSC-A parameter on the y-axis.
Click on Rectangle and create a "relaxed" gate to include all single live cells in that graph. Double-click in the center of the relaxed gate to display the cells in a dot plot with CD3 on the x-axis and CD8 on the y-axis.
Use the appropriate axis scale for visualization in the graph window. If necessary, modify the x and y scale by clicking on the box indicated with a T close to each axis. Choose Customize Axis Function and modify Extra Neg Decades, Ancho Basis, and Positive Decades as necessary.
Select the CD3-positive, CD8-positive cells by clicking on Polygon. Double-click in the center of the CD3-positive, CD8-positive gate to display the cells in a dot plot with Tetr-gag on the x-axis and Pent-gag on the y-axis. Select the antigen-specific CD8 T cells by clicking on Polygon. Double-click in the center of the gag-specific gate to display the cells in a dot plot with DNA-A on the x-axis and Ki67 on the y-axis. Select the cells in the different cell cycle phases by clicking on Quad in the Gating Tools section of the graph window.
