Cd45 til microbead kit

The tumors from PD-1 Ab-treated, PD-1KO, and PD-1cKO (day 18 post-tumor implant) mice and respective WT controls were collected and digested using the tumor dissociation kit (Miltenyi Biotech), followed by isolation of CD45+ TILs using the CD45 (TIL) MicroBead kit (Miltenyi Biotech). The cells were counted before proceeding for cell surface staining.

The tumors were collected from mice and digested using the tumor dissociation kit (Miltenyi Biotech), followed by isolation of CD45⁺ TILs using the CD45 (TIL) MicroBead kit (Miltenyi Biotech). Staining of cells with CisPt 194 was followed by incubation with Fc block and, subsequently, the panel of antibodies for cell surface markers. To perform intracellular staining, the fixation/permeabilization buffer was used (BD Biosciences). The cells were permeabilized for 30 mins at 4°C and stored overnight in the cell staining medium, and subsequently, bar coding was performed using the barcoding reagent according to manufacturer’s instructions (Fluidigm). Next, intracellular staining for cytokines or transcription factors was performed by incubating the cells in the respective antibody mixture. Finally, the cells were incubated overnight in permeabilization buffer and iridium nucleic acid intercalator (Fluidigm). Prior to acquisition, cells were washed and then diluted to 5 × 10⁶ cells/ml in dH₂O containing 10% (v/v) EQ Four Element Calibration Beads (Fluidigm) and filtered. Cells were acquired at a rate of 400 cells/s using a Helios mass cytometer (Fluidigm). Flow Cytometry Standard (FCS) files were normalized to EQ bead signal and debarcoded using Fluidigm de barcoder algorithm. Files were analyzed using Cytobank CytoF v6.7 (Fluidigm).