For mouse experiments, tissues were processed, single cell suspensions obtained, and cells were stained as described (Wherry et al., 2003 (link)). Mouse cells were stained with LIVE/DEAD cell stain (Invitrogen) and antibodies targeting surface or intracellular proteins. Intracellular cytokine staining was performed after 5 hrs ex vivo stimulation with GP33-41 peptide in the presence of GolgiPlug, GolgiStop and anti-CD107a. After stimulation, cells were stained with surface antibodies, followed by fixation with Fixation/Permeabilization Buffer and then stained with intracellular antibodies for TNF, IFN-γ and MIP1α using Permeabilization Wash Buffer according to manufacturer’s instructions. Flow cytometry was performed with an LSRII. Cell sorting experiments were performed with a BD-Aria sorter, with 70-micron nozzle and a 4°C circulating cool-down system for sequencing, western and TIDE assays.
For sorting RV+ cells optimized sorting in the transfer experiments, the BD Aria Sorter was set at 37°C and 100-micron nozzle, with a flow rate lower than 3.0. 3 X 106 Cells are concentrated in 300ul 10% complete RPMI with 100 U/mL recombinant human IL-2 during sorting. 37°C pre-warmed collection tubes with 10% complete RPMI (100 U/ml IL-2) are used. Sorted cells are washed by 37°C warm pure RPMI before transferring into recipients.
For sorting RV+ cells optimized sorting in the transfer experiments, the BD Aria Sorter was set at 37°C and 100-micron nozzle, with a flow rate lower than 3.0. 3 X 106 Cells are concentrated in 300ul 10% complete RPMI with 100 U/mL recombinant human IL-2 during sorting. 37°C pre-warmed collection tubes with 10% complete RPMI (100 U/ml IL-2) are used. Sorted cells are washed by 37°C warm pure RPMI before transferring into recipients.