Multicolor Flow Cytometry Assay for Immune Profiling

Tumor cell suspensions (4 × 10⁶) prepared by mechanical and enzymatic dissociation^{16 (link)} were stained in 96-wells round bottom plates with live/dead staining (Blue fluorescent reactive dye, #L34962 Invitrogen) during 20 min at room temperature. For flow-cytometry analysis and sorting, Fc receptors were blocked with anti-FcR (anti-CD16 and CD32, at 5 µg/ml, Biolegend #101339). After two washes in PBS 2% FCS, cells were stained with the following antibodies (all used at 1:100): anti-CD11b-BV421 (#562605), CD64-APC (#558539), CD11c-PeCy7 (#557401), TCRβ-BV605 (#562840) and CD4-BV711 (#563050) all purchased from BD Pharmingen; anti-CD45-AF700 (#103128), Ly6C-APCCy7 (#128025), Ly6G-BV510 (#127633), F4/80 BV650 (#123149), CD206-PE (#141706), IA/IE-BV785 (#107645) all purchased from Biolegend, and CD8-PerCPef710 (#46-0081-82) purchased from eBioscience. After washing, cells were fixed in 1% PFA, stored at 4 °C, and acquired the next day on LSR II or FORTESSA (BD Bioscience). For detection of phosphorylated proteins, cell suspensions were stimulated 3 h with DMXAA 250 µg/ml, fixed immediately in PFA 4%, permeabilized with frozen methanol 90%, stained overnight with 1:100 pTBK1-PE (#13498) antibodies (purchased from Cell signaling), then washed and further stained for multicolor flow cytometry.