Anti mouse cd8α pe

IFN-γ release of induced CD8⁺ T cells was measured by the intracellular cytokine staining assay according to our former study [31 (link), 34 (link)]. Briefly, the induced CTLs (1 × 10⁶) and T2A2 cells loaded with/without corresponding peptide (1 × 10⁶) were incubated by adding protein transport inhibitor (containing brefeldin A) for 5 hours at 37°C. Afterward, cells were harvested, transferred to 1.5 mL EP tubes, and washed by PBS (contain 2% FBS). Cells were then stained for cell surface markers (anti-human CD3 PerCP-eFlour (Clone: OKT3, 46-0037-42, eBioscience, USA)/anti-mouse CD3 PerCP-eFlour710 (Clone: 17A2, 46-0032-80, eBioscience, USA) and anti-human CD8α APC (Clone: SK1, 17-0087-42, eBioscience, USA)/anti-mouse CD8α PE (Clone: 53-6.7, 12-0081-82, eBioscience, USA)) for 30 min at 4°C. Afterward, the cells were fixed by fixation buffer for another 30 min at room temperature and washed twice with permeabilization wash buffer. Anti-human IFN-γ PE (Clone: 4S.B3, 12-7319-42, eBioscience, USA) or anti-mouse IFN-γ APC (Clone: XMG1.2, 17-7311-81, eBioscience, USA) was added to those tubes for intracellular staining for 30 min at 4°C. After being washed twice with permeabilization wash buffer, the IFN-γ release of CD8⁺ T cells was analyzed by flow cytometry (FACS Calibur, BD Bioscience, USA).

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.

For the T2A2 cell-binding assay, monoclonal antibody HLA-A2 PE-Cyanine7 (clone: BB7.2, # 343314) was purchased from BioLegend (San Diego, CA, USA). For intracellular cytokine staining assay, monoclonal antibody anti-human CD3 eflour450 (clone: OKT3, #48-0037), anti-human CD8α PerCP-eFlour 710 (clone: SK1, #46-0087), anti-human IFN-γ PE (clone: 4S.B3, #12-7319), anti-human granzyme B PE (clone: GB11, #12-8899), anti-mouse CD3 PerCP-eFlour 710 (clone: 17A2, #46-0032), anti-mouse CD8α PE (clone: 53-6.7, #12-0081), anti-mouse IFN-γ APC (clone: XMG1.2, #17-7311), and anti-mouse granzyme B eFlour 450 (clone: NGZB, #48-8898) were purchased from eBioscience (San Diego, CA, USA). To detect the mature cells (DCs), anti-human CD80 PerCP-eFlour 710 (clone: 2D10.4, #46-0809), anti-human CD86 PE (clone: IT2.2, #12-0869), and anti-human HLA-DR APC (clone: LN3, #17-9956) were purchased from eBioscience (USA).