Lsr 2 x 20 fortessa

Cell culture supernatants were harvested and frozen at −80 °C for later quantification of TNF, IL-2, IFN-γ, IL-10, IL-6, IL-4, and IL-17A by cytometric bead array (Human Th1/Th2/Th17 Kit, BD Biosciences) according to the manufacturer’s instructions. CBA data were acquired on an LSR II X-20 Fortessa (BD Biosciences) and analyzed using the LEGENDplex software (BioLegend). The limits of detection for all assays was 20 pg/ml. Sample values below the limit of detection (LOD) were set to LOD/2.

For characterization of TIL, mice were euthanized at the days specified in the results section. Tongue and flank tumors were collected and digested as previously described [10 (link)]. Purified leukocytes were stained for multi-parametric flow cytometry analysis with a 16-color antibody panel. Cells were blocked with mouse Fc-block, stained with surface markers, fixed and permeabilized with the FoxP3 Fix/Perm Kit (eBioscience, Waltham, MA) followed by staining for intracellular markers. Samples were run in an LSR-II X-20 Fortessa (BD Biosciences, San Jose, CA) at the South Campus Flow Cytometry Core, MD Anderson Cancer Center (Houston, TX) and analyzed using FlowJo version 10 (Flowjo LLC, Ashland, OR). The live/dead fixable aqua dye (Thermo Scientific, Waltham, MA) was used to gate out dead cells and to include only live cells for analysis. Live leukocyte gate was set based on forward and side scatter to include both lymphocytes and larger myeloid cells. Tregs were identified based on CD4⁺Foxp3⁺ expression within the CD3⁺ gate. From CD3⁻ gate, CD11b⁺Gr-1⁺ cells were identified as total MDSC population. The ratio of CD8⁺ T cells to Tregs or MDSC were calculated by dividing the percentage of CD8⁺ T cells with that of CD4⁺Foxp3⁺ or CD11b⁺Gr-1⁺ cells.

Plasma was diluted 1:2 and 1:4 for assessment of cytokines using the human soluble protein cytometric bead array flex sets (BD Biosciences) according to manufacturer’s instructions. Cytometric bead array data were acquired on a BD LSR II X-20 Fortessa and analyzed using the FCAP Array Software. The following 18 cytokines were quantified: IL-1α, IL-1β, IL-6, IFNα, TNFα, MIP-1α, MCP-1, IL-8, RANTES, IL-12p70, IL-10, IL-2, IL-5, IL-5, IL-9, IL-13, IFNγ, and IL-17A. All cytokines except for IL-8, MCP-1, and RANTES fell below the limit of detection of the assay at both dilutions and were excluded from future analysis. Results are reported in pg/mL and plotted using Prism version 8.0.0.