Brilliant violet 605 anti mouse cd45

The antibodies used for flow cytometry were Brilliant Violet 421™ anti-mouse CD8α (Biolegend; Clone 53-6.7), APC/Cy7 anti-mouse CD8β Antibody (Biolegend; YTS156.7.7), Brilliant Violet 605™ anti-mouse CD45 (Biolegend; 30-F11), PerCP/Cy5.5 anti-mouse CD103 (Biolegend; 2E7), BV421 anti-Mouse CD103 (BD Biosciences; M290), PE-Cyanine7 anti-human/mouse CD44 (Tonbo; IM7), PE anti-mouse CD279/PD-1 (Tonbo; J43.l), FITC anti-mouse CD279/PD-1 (eBioscience; RMP1-30), FITC, anti-mouse CD69 (BD Biosciences; H1.2F3), and FITC anti-mouse CD90.1 (eBioscience; HIS51). Allophycocyanin labeled H-2D^b/VP2_121–130, H-2K^b/SIYR, and H-2K^b/OVA_257–264 tetramers were kindly provided by Dr. Aaron Johnson (Mayo Clinic, Rochester, MN, USA) and were generated using previously described methods (17 (link), 20 (link)). Anti-PD1 hamster monoclonal antibody (G4) was purified and dosed as previously described (21 (link)). Animals were treated with 200 µg of anti-PD-1 or isotype IgG on day three post-infection with TMEV-OVA8. The antibody used for in vivo intravascular labeling of CD8⁺ T-cells was PE anti-mouse CD8α (BD Biosciences; 53-6.7). Intravascular labeling of peripheral blood lymphocytes and CNS-IL was performed using previously described methods (22 (link)).

Concanavalin A (Con A) and lipopolysaccharide (LPS) were purchased from ApexBio Technology Co., Ltd. and Sigma-Aldrich (Shanghai) Trading Co., Ltd., respectively. Mouse ELISA kits for IL-4 (20220111-20186A), IFN-γ (20220111-20140A), TNF-α (20220111-20852A), MCP-1 (20220111-20248A), IL-1 β (20220111-20174A), and IL-12 (20220111-20166A) were purchased from Shanghai Enzyme Linked Biotechnology Co., Ltd. The following flow cytometry antibodies were purchased: Brilliant Violet 605™ anti-Mouse CD45 (BioLegend), BV421 Rat anti-mouse CD25 (BD); BV510 Hamster anti-mouse CD3e (BD); mCD8-R208-APC (Sino Biological); mCD4-R711-FITC (Sino Biological); FITC anti-Mouse MHC-II (I-A/I-E) (eBioscience); BV421 Hamster anti-Mouse CD80 (BD); PE Rat anti-Mouse CD86 (BD); and BV786 Hamster ant-mouse CD11c (BD).

Single cell suspensions of PDX tumor cells were stained with Brilliant Violet 605 anti-mouse CD45 (Biolegend #103139); BB515/FITC anti-mouse CD11b (BD Bioscience #564454); MHCII-APC (MBL International Corporation FP20868002); PE anti-mouse Ly6C (BD Bioscience #560592); and BV421 anti-mouse Ly6G (BD Bioscience #562737). Cells were counterstained with the live/dead fixable near-IR cell death marker (Thermo Fischer Scientific). The cells were analyzed on a BD FACSAria™ III (BD bioscience). Data was analyzed using FlowJo software (version 10, FlowJo).
For cell sorting experiments, single cell suspensions were stained as described above, and the two populations (CD45⁺CD11b⁺MHCII⁻LY6C⁻Ly6G⁻) and (CD45⁺CD11b⁺MHCII⁻LY6C⁻Ly6G⁺) were sorted in TRI reagent (Sigma-Aldrich) using a BD FACSAria™ III (BD bioscience).