Lsr 2

Single cell suspensions of livers were washed in PBS prior to viability staining (Zombie Near-InfraRed) and staining for surface proteins for 30 minutes. Excess antibody was washed out with PBS prior to sample fixation. All antibodies were used at a 1:200 dilution, except F4/80-PE-Cy7 which was used at 1:150, for 30 minutes on ice. Samples were analyzed using a BD LSR-II (4 lasers) and a Cytek Aurora (4 lasers). Antibodies used for flow cytometry included the following: BV421 anti-mouse F4/80 (BD, 100433), eFluor 506 anti-mouse CD45 (Invitrogen, 69-0451-82), FITC anti-mouse Ly6G (BD, 553127), PerCP/Cyanine5.5 anti-mouse CD4 (Biolegend, 12-0114-81), PE anti-mouse CD11c (Invitrogen, 25-0032-82), PE Cyanine 7 anti-mouse CD3 (Invitrogen, 565411), APC anti-mouse CD11b (Biolegend, 101211) and live/dead near-IR dead cell stain kit (Invitrogen, L34975).

Mice were sacrificed, TDLN, spleen and tumors were removed. We placed the spleens and lymph nodes between the frosted surfaces of two glass slides and applied force to disrupt these organs to release immune cells, spleen was need to treated with ACK lysis buffer to remove red blood cells. Single-cell suspensions were filtered through a 40-μm cell strainer, washed, and resuspended in 1%FBS HBS for analysis. Tumor were cut into small pieces, digested in serum free RPMI with 0.25 mg/ml Liberase TL (Roche) and 0.33 mg/ml Dnase 1 (Sigma) in 37° for 30 min. Single-cell suspensions were filtered through a 40-μm cell strainer, washed, and resuspended in 1%FBS HBS for staining. For IFN-γ or Granzyme B staining, tumor cells were stimulated with leukocyte activation cocktail (Biolegend) for 6 h, then stained surface marker and intracellular markers. by the standard staining protocol described before (Chen et al., 2020 (link)). Flow cytometry analysis were applied to LSRII or Aurora (Cytek Biosciences) and analyzed by using Flowjo software (BD).