Brilliant violet 711 cd4

BBN963 cells (5 × 10⁶) or MB49 cells (1 × 10⁶) were subcutaneously injected into the right and left flank of each C57BL/6 mice. Right side is designed as primary tumor and treated with light and PNP or anti-PD-1 plus PNP groups while the left side is designed as secondary site with no light treatment.
Mice were treated with PBS, anti-PD-1, PNP+L, anti-PD-1+PNP+L for two weeks. Left tumors were collected to make single cell suspension. The harvested cells were analyzed by flow cytometry or cytometry by time of flight (CyTOF) and/or immunofluorescence staining. Antibodies for the tumor tissue are Zombie Aqua (Biolegend, 423102, 1:400), APC-CD3 (Biolegend, 100312, 1:100), Brilliant Violet 711-CD4 (Biolegend, 100550, 1:100), PE/Cy7-CD8a (Biolegend, 100722, 1:100), BUV395-CD45(BD, 564279, 1:100). CyTOF analysis was performed at the Human Immune Monitoring Center at Stanford University. Tumor samples were stained with antibody-polymer conjugate cocktail (Maxpar® Mouse Sp/LN Phenotyping Panel Kit, 16 Marker, #201306 Fluidigm Corporation; each at 1 μL/test) following the manual procedure. ELISA kits (R&D Systems, Mouse Luminex Discovery Assay, LXSAMSM) were used to test cytokine levels.
Immunohistochemical (IHC) staining was performed according to the manufacturer’s instruction (BioGenex). IHC primary antibodies against CD8a (CST, 98941S, 1:100), Ki-67 (Abcam, ab279653, 1:1000).