Assessment of immune microenvironment by flow cytometry

To assess the tumor immune cell infiltration, the tumors were harvested, processed, and stained as described previously.24 25 Briefly, tumors were digested at 37°C for an hour in a digestion cocktail buffer prepared in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich) containing DNase I (20 U/mL; Sigma-Aldrich), Collagenase I (1 mg/mL; EMD Millipore) and Collagenase IV (250 U/mL; Worthington Biochemical Corporation), followed by mechanical disruption to form single cell suspensions. Single cell suspensions were also prepared from blood, spleen, and inguinal lymph nodes with spleen and blood needing an additional lysis of the red blood cells (RBC) using Invitrogen’s RBC lysis buffer. Cells were blocked with the anti-mouse CD16/CD32 Fc block (BD Biosciences) and then separately stained for the extracellular T-cell and the myeloid cell antibodies panel with details of all the antibodies included in online supplemental table S1. For intracellular antibody staining cells were first fixed and permeabilized (eBioscience Intracellular Fixation and Permeabilization Buffer Set; Invitrogen). Data for both antibody panels were acquired on LSRFortessa (BD Biosciences) flow cytometer and analyzed using FlowJo V.10 software (FlowJo). Gating strategies are outlined in online supplemental figure S1.

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