To begin, obtain a six to eight week old CD 45.1 positive SMARTA mouse and inject it with 200 micrograms of LCMV GP 61-77 peptide in 100 microliters of RPMI medium intravenously. After acquiring the splenocytes from the mouse, suspend them in 2%RPMI to achieve a cell density of one times 10 to the eighT-cells per milliliter and place the tube with cells on ice. Dispense 50 microliters of cell suspension along with 150 microliters of staining buffer to each well of a round-bottom 96-well plate.
Centrifuge the 96-well plate at 800 G for one minute at four degrees Celsius and carefully decant the supernatant. Vortex the plate and add 200 microliters of staining buffer to each well. After pelleting the cells, resuspend and incubate them with surface antibody cocktail on ice for 30 minutes in the dark.
After washing two times, resuspend the cells in 200 microliters of staining buffer and transfer them into a flow cytometry tube. Perform flow cytometry analysis to check the activation status of SMARTA CD4 positive T-cells. Then seed one times 10 the six SMARTA CD4 positive T-cells per well into a 24 well plate.
Spin down the cells at 800 G for three minutes at four degrees Celsius and carefully remove the supernatant medium. Next, add one milliliter of retrovirus medium and eight micrograms of polybrene to each well. Spin transduce the cells at 800 G for two hours at 37 degrees Celsius.
After discarding the retrovirus medium, incubate the cells with one milliliter of prewarmed 10%RPMI supplemented with interleukin 2. Transfer the cell suspension into a 15 milliliter conical tube and centrifuge it. After discarding the supernatant, resuspend the cells with 2%RPMI to achieve a cell density of one times 10 to the eighth cells per milliliter.
For assessing the transduction efficiency, aliquot 50 microliters of cell suspension into a round bottom 96-well plate. Incubate them on ice with the surface antibody cocktail including CD4, V alpha two, and vector tag-associated antibody. Wash the cells and perform flow cytometry as demonstrated earlier.
Then inject one times 10 of the six CD 45.1 positive SMARTA CD4 positive T-cells to a C57BL/6 recipient mouse intravenously followed by one times 10 to the six plaque forming units of LCMV Armstrong the next day. After acquiring the splenocytes from the mouse, stain them to check the desired markers. To detect transcriptional factors, incubate the cells with 200 microliters of 2%paraformaldehyde at room temperature for 20 minutes in the dark.
Finally, perform flow cytometry to detect factors at the early acute LCMV infection stage. On day three after infection, a balanced bifurcation of follicular helper T-cells and T-helper 1 cells Among MIGR1 SMARTA CD4 positive T-cells was found. A predominant follicular helper T-cell directed differentiation and enhanced BCL6 expression levels were observed in MIGR1 BCL6 SMARTA CD4 positive T-cells.
