Apc anti mouse cd8a antibody

A total of 60 µL blood was collected and stained with 10 µL E7-specific or p15E-specific tetramer (both provided by NIH tetramer core facility) to a final 500 times dilution for 1 hour on ice with constant shaking. Aqua fluorescent reactive dye for live and dead staining (Invitrogen, catalog no. L34957) in a final 500 times dilution, FITC anti-mouse CD4 Antibody (BioLegend, catalog no. 100406) and APC anti-mouse CD8a Antibody (BioLegend, catalog no. 100712) in a final 200 times dilution was added to the testing samples to a total volume of 100 µL and incubated for 30 minutes on ice with constant shaking. After one wash with cold PBS and centrifugation at 1,350 rpm for 3 minutes, the cells were resuspended in 200 µL FACS buffer and ready for examination by using BD LSRFortessa X-20 cytometer. Data were processed by Flowjo (version 10).

Mouse spleens were harvested on the 56th day after the first vaccination, and single-cell suspensions were prepared by processing them through a 70 μm cell strainer ( BD Biosciences, San Jose, CA, USA). Red blood cells presenting in the splenocyte suspensions were lysed using Red Blood Cell Lysis Buffer (Beyotime, Shanghai, China). The cells were inoculated into 96-well plates, and 20 μM antigenic peptides (M2e+HA2) were added to each well. After 1 h of incubation, we added the Brefeldin A Solution (1X) to each well and incubated them for another 5 h. Then, we washed the cells once with PBS buffer, added FITC anti-mouse CD3ϵ, PE/Cy7 anti-mouse CD4, and the APC anti-mouse CD8a antibody simultaneously (BioLegend, San Diego, CA, United States). Next, we incubated them at 4°C for 45 min and washed them with PBS twice, after which, we added the Cytofix/Cytoperm™ Solution and incubated them at 4°C away from light for another 20 min. Then, we washed the cells twice by using the BD Perm/Wash™ Buffer (BD, San Jose, CA, United States) and divided each tube of cells equally into 3 tubes; next, we added the PE anti-mouse IL-2, IL-4, or IFN-γ antibody, respectively, and incubated them for 1 h at 4°C away from light. After being washed twice, the stained cells were put into flow cytometry (Beckman Coulter, Miami, FL, United States) for detection.

AT-infiltrating stromal cells were stained with FITC anti-mouse CD3ε antibody, PE anti-mouse CD4 antibody, PE/Cy7 anti-mouse CD25 antibody, APC anti-mouse CD8a antibody, FITC anti-mouse CD127 antibody, PerCP/Cy5.5 anti-mouse CD4 antibody, APC anti-mouse CD25 antibody (these antibodies were purchased from Biolegend, San Diego, CA, USA), PE anti-mouse/Foxp3 antibody (Invitrogen, Carlsbad, CA, USA), PE anti-mouse CD11b antibody (Biolegend), PE/Cy7 anti-mouse Ly-6C antibody (Biolegend, San Diego, CA, USA) and APC anti-toll-like receptor 4 (TLR4) antibody (Invitrogen, Waltham, MA, USA), and analyzed using an Accuri C6 (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).