After enriching the lung sample for T cells, add one milliliter of 10 millimolar dasatinib to each sample. Vortex the mixture and incubate for five minutes at room temperature. Add PE or APC conjugated peptide MHC tetramer to a final concentration of 10 nanomolar.
After vortexing the mixture, incubate it in the dark for one hour at room temperature. While the sample is stained with peptide MHC tetramers, prepare a master mix of antibodies to stain the cell surface markers with 15 minutes remaining in the tetramer staining incubation period, add the surface antibody master mix at a 1 to 100 dilution. Add 50, 000 flow cytometry count beads and cold sorter buffer to reach a final volume of approximately five milliliters, then add cold sorter buffer to a volume of 15 milliliters and centrifuge the tube at 400 x g for five minutes at four degrees Celsius.
Carefully aspirate the supernatant, leaving approximately 100 microliters of the supernatant and the cell pellet. Now Resuspend the sample with an additional 200 microliters of sorter buffer and transfer it to a five milliliter FACS tube.
