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4 protocols using anti il 2 bv510

1

Multicolor Flow Cytometry Analysis of Tumor-Infiltrating Lymphocytes

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Nine days after the treatment of MC38 tumours with indicated compounds, tumor infiltrating lymphocytes (TILs) were isolated and stained. For stimulation, cells were incubated with cell stimulation cocktail (PMA and Ionomycin) plus protein transport inhibitor (Brefeldin A and Menesin) for 4 hrs. Cells were analysed by multicolour flow cytometry (BD LSR Fortessa X-20). Antibodies: anti-CD45 BV605 (#103140, Biolegend); anti-CD3 BV875 (#100355, Biolegend); anti-CD4 BV650 (#100469, Biolegend); anti-CD8 (#100784, Biolegend); anti-FOXP3 PerCP-Cy5.5 (#563902, BD); anti-TNFα APC (#506308, Biolegend); anti-IFNγ FITC (#505806, Biolegend); anti-IL-2 BV510 (#503833, Biolegend); anti-IL10 PerCP-Cy5.5 (#505028, Biolegend). All analyses were performed using FlowJo_V10.6.1 software (Tree Star). Gating strategy: gate cells exclude dead cells and debris based on cells size, then gate live cells based on Live-Dead NIR negative cells, then gate CD45+ cells, then gate CD45+CD3+ cells, then gate CD45+CD3+CD8+ cells and CD45+CD3+CD4+ cells. Cytokine expression was determined in the populations of CD45+CD3+cells.
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2

Multi-parameter Flow Cytometry Characterization of TCR-engineered T Cells

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TCR engineered cells were incubated with the antigens for various times, washed and rested for up to 72 h and then re-stimulated with 1× phorbol 12-myristate 13-acetate (PMA), Ionomycin, brefeldin A and monensin cocktail (eBioscience) for 5–7 h. Cells were harvested and surfaced stained with anti-human CD4 PeCy5 (Biolegend) in FACS buffer (homemade). Fixable viability dye efluor 780 (eBioscience) was added and cells were incubated for 30 min on ice, washed and then incubated in 1× fixation/permeabilization buffer (eBioscience) overnight at 4 °C. After fixation/permeabilization, cells were stained for cytokines and transcriptions factors in 1× permeabilization buffer(eBioscience) overnight at 4 °C. Cytokines were stained using anti-IFN-γ PeCy7, anti-IL-2 BV510, anti-IL-4 PE (all from Biolegend) and anti-IL-17A PerCPCy5.5 (eBioscience). The transcription factors were stained using anti-T-bet PeCy7, anti-GATA3 PE (both Biolegend) and anti-RORγt APC (eBioscience). All data were acquired using a BD LSR II flow cytometer and analyzed using Flowjo software (Flowjo, LLC).
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3

Multicolor Flow Cytometry Analysis of Tumor-Infiltrating Lymphocytes

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Nine days after the treatment of MC38 tumours with indicated compounds, tumor infiltrating lymphocytes (TILs) were isolated and stained. For stimulation, cells were incubated with cell stimulation cocktail (PMA and Ionomycin) plus protein transport inhibitor (Brefeldin A and Menesin) for 4 hrs. Cells were analysed by multicolour flow cytometry (BD LSR Fortessa X-20). Antibodies: anti-CD45 BV605 (#103140, Biolegend); anti-CD3 BV875 (#100355, Biolegend); anti-CD4 BV650 (#100469, Biolegend); anti-CD8 (#100784, Biolegend); anti-FOXP3 PerCP-Cy5.5 (#563902, BD); anti-TNFα APC (#506308, Biolegend); anti-IFNγ FITC (#505806, Biolegend); anti-IL-2 BV510 (#503833, Biolegend); anti-IL10 PerCP-Cy5.5 (#505028, Biolegend). All analyses were performed using FlowJo_V10.6.1 software (Tree Star). Gating strategy: gate cells exclude dead cells and debris based on cells size, then gate live cells based on Live-Dead NIR negative cells, then gate CD45+ cells, then gate CD45+CD3+ cells, then gate CD45+CD3+CD8+ cells and CD45+CD3+CD4+ cells. Cytokine expression was determined in the populations of CD45+CD3+cells.
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4

Multiparameter Flow Cytometry Analysis

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Cells were stimulated (3 h) with phorbol-12-myristate 13-acetate (PMA; 50 ng ml−1, Sigma-Aldrich, P1585), ionomycin (1 μg ml−1, Sigma-Aldrich, I3909) and Golgi Stop (1:100, BD Biosciences, 554724). The cells were then fixed and permeabilized (1:100, BD Biosciences, 554714) and stained with anti-IFNγ–APC (1:100, BioLegend, 505810), anti-IFNγ–FITC (1:100, BioLegend, 505806), anti-TNF–BV510 (1:100, BioLegend, 506339), anti-TNF–BV5711 (1:100, BioLegend, 506349), anti-TNF–PE (1:100, BioLegend, 506306), anti-IL2–Pecy7 (1:100, BioLegend, 503832), anti-IL-2–Pacific Blue (1:100, BioLegend, 503820), anti-IL-2–BV510 (1:100, BioLegend, 503833), and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).
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