This method will enable the characterization of different immune cells in the same states of disease of individuals with IM, which will expand our understanding of the mechanism of EBV infection. Demonstrating the procedure will be Linlin Zhang, a doctor from our laboratory. To begin, take the PBMCs isolated previously in a 1.5 milliliter microcentrifuge tube, and spin it at 300 G for 10 minutes at room temperature.
Discard the supernatant, and re-suspend the cells with 80 microliters of the prepared buffer. Next, add 20 microliters of CD 14 microbeads to the cell suspension, mixed well by pipetting, and incubate the tube for 15 minutes on ice. Then wash the PBMCs using one milliliter of buffer and centrifuge at 300 G for 10 minutes at room temperature.
Discard the entire supernatant, and re-suspend the cells with 500 microliters of the buffer. For magnetic separation, place a column on the magnetic bead separator, and prime the column by washing it with three milliliters of the buffer. Next, add the cell suspension to the column.
Allow it to pass through, and collect the unlabeled cells into a 15 milliliter centrifuge tube. Wash the column three times with three milliliters of the buffer, and collect the entire effluent in a 15 milliliter centrifuge tube. Now, place the column in a new 15 milliliters centrifuge tube, and add five milliliters of buffer to the column.
Push the plunger firmly into the column to flush the magnetically labeled cells into the tube. Centrifuge the magnetically labeled cells at 300 G for five minutes. After removing the supernatant, re-suspend the cells in 500 microliters of PBS, and transfer the cell suspension to a 1.5 milliliter microcentrifuge tube for subsequent transcriptome sequencing.
Pellet the collected unlabeled cells by centrifuging at 300 G for five minutes, and re-suspend the cells in 100 microliters of PBS in a 1.5 milliliter microcentrifuge tube. Then, add two microliters of each labeled antibody to the cell suspension, and incubate on ice for 30 minutes, protected from light. Next, aliquot 100 microliters of the PBMC cell suspension in 1.5 milliliter microcentrifuge tubes to set up a negative control sample CD 3, CD 4, CD 8, and CD 19, single staining samples, and a CD 56, or CD 16 staining sample.
Then add two microliters of the corresponding fluorescently labeled antibody to each tube, and incubate on ice for 30 minutes, protected from light. After incubation, wash all cells with PBS as demonstrated previously, and re-suspend in 500 microliters of PBS. Open the cell sorting system, and run the power on program.
Then, install the 85 micrometer nozzle, and open the sorting stream. Set the sorting voltage to 4, 500 volts, and the frequency to 47. Set up the gap to 8 in the break-off window, and turn on the sweet spot automatic sorting mode to determine the droplet amplitude value automatically.
Finally, adjust the droplet delay to 30.31 in the side stream window. Using a forward versus side scatter dot plot, draw the polygon gate P1 to identify the intact lymphocyte population. Next, using a forward scatter area versus height dot plot, draw the polygon gate P2 to identify the single cells, and exclude doublets.
Then using a CD 19 FITC area versus CD 3 per CPCY 5.5 area dot plot, Draw the rectangular gate P3 to select CD 3 positive T-cells, and CD 19 positive B-cells. Next, using a CD 4 APCCY 7 area versus CD 8 PE area dot plot, draw a rectangular gate to select CD 4, and CD 8 positive T-cells. Finally, using a side scatter area versus CD 56 or CD 16 APC area dot plot, draw a rectangular gate to select CD 56, or CD 16 positive natural killer cells.
For the negative control tube, click load in the acquisition dashboard, and select the cytometer settings in the software. In the inspector window, click the parameters tab, and adjust the voltages of forward scatter, side scatter, and different fluorescent dyes. After loading the single stained tubes into the cytometer, click load in the acquisition dashboard, then click the compensation tab to adjust the compensation, as described in the text.
Vortex the cell suspension gently, before loading the tube into the cytometer. Add 200 microliters of FBS to four collection flow tubes, and vortex the tubes. Place them in the cytometer collection chamber.
Collect the different types of cells separately in the four flow tubes. Spin down the separated cells at 300 G for five minutes, and add 200 microliters of RNA isolation reagent to the cell palette for transcriptome sequencing. In the present study, immune cells subpopulations from peripheral blood of different samples were sorted out efficiently by combining the techniques of immunomagnetic beads, and fluorescence activated cell sorting.
Importantly in this protocol, the cell sorting step has been optimized to improve cell viability. The sorted immune cells showed an increased proportion of CD 3, CD 8 positive T-cells in patients with infectious mononucleosis compared to both the healthy Epstein-Barr virus carriers, and uninfected samples. In contrast, decreased proportions of CD 3, CD 4 positive T-cells, and CD 19 positive B-cells were observed in patients with infectious mononucleosis compared with healthy EBV carriers, and EBV uninfected children.
Additionally, decreased proportions of CD 56, or CD 16 positive natural killer cells were observed in patients with infectious mononucleosis compared with EBV uninfected children. Maintaining cell viability is vital for subsequent experiment. Keeping cells on ice, or in the refrigerator during the sorting process is beneficial for cell viability.
