Evaluating CTL Response to MTA1 Stimulation

To test the protein MTA1_(1–283) stimulation of CTL response in vivo, IFN-γ release of induced CTLs was determined by the intracellular cytokine staining assay according to our former study [32 (link)]. In brief, on day 14, spleen lymphocytes were harvested and stimulated by peptide pool (10 μg/ml) in vitro for another 6 days. Then, restimulated splenocytes were incubated with peptide-loaded T2 cells or T2 cells only for 3 h. Subsequently, protein transport inhibitor cocktail was added to splenocytes for another 4 h. Then, those cells were stained with Anti-mouse CD3 PerCP-eFluor® 710 (clone: 17A2, eBioscience) and Anti-mouse CD8α PE (clone: 53–6.7, eBioscience) at 4°C for 30 min. And those cells were fixed by IC fixation buffer (cat no: 00-8222, eBioscience) at room temperature for another 30 min; then those cells were washed twice by permeabilization buffer (cat no: 00-8333-56, eBioscience). After, those cells were incubated with Anti-mouse IFN-γ APC (clone: XMG1.2, eBioscience) at 4°C for 30 min. Those cells were washed twice by permeabilization buffer and analyzed by flow cytometry.