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3 protocols using pe conjugated anti il 2

1

Immune Cell Profiling by Flow Cytometry

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The immune cell populations were subjected to surface staining for flow cytometry analysis. The used antibodies are as follows: PE‐conjugated anti‐CD3ε (Biolegend), PerCP‐conjugated anti‐CD4 (BD Pharmingen), PerCP‐conjugated anti‐CD8α (BD Pharmingen), FITC‐conjugated anti‐NK1.1 (Biolegend), FITC‐conjugated anti‐CD11b (Biolegend), PE‐conjugated anti‐CD103 (Biolegend), APC‐conjugated anti‐CD11c (BD Pharmingen), PerCP‐conjugated anti‐F4/80 (Biolegend), or FITC‐conjugated anti‐Gr‐1 (Biolegend).
For analyzing cytokine secretion, splenocytes were inoculated into a 12‐well plate at 4 × 106/well and incubated with 10 μg/mL CAIX protein at 37°C and 5% CO2 for 3 days, then added 500 ng/mL Ionomycin (Sigma‐Aldrich) and 50 ng/mL PMA (Sigma‐Aldrich) and 5 ng/mL Brefeldin A (BFA, eBioscience) to incubate for 5 hours. Then cells were collected and operated extracellular staining with anti‐mouse PerCP‐conjugated anti‐CD8α and intracellular staining with following anti‐mouse antibodies: APC‐conjugated anti‐IFN‐γ (BD Pharmingen), PE‐conjugated anti‐IL‐2 (BD Pharmingen), FITC‐conjugated anti‐TNF‐α (BD Pharmingen). The data acquired from BD FACSCanto II (BD Biosciences) in FACSDiva software were analyzed by FlowJo software (Tree Star Inc).
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2

Murine LAIR-1 Antibody Characterization

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Antibodies used for this study included: monoclonal anti-murine LAIR-1 antibodies, (Affymetrix/eBioscience, San Diego, Ca) and Armenian hamster IgG isotype control for the anti LAIR-1 (Biolegend, San Diego, Ca). The Abs used for flow cytometry included: PacBlue-conjugated anti-CD4, PE-conjugated anti-IL-2, APC-conjugated anti-IFN-γ, FITC-conjugated anti-CD8, APC-conjugated anti-CD19, FITC-conjugated anti-CD11c, APC-conjugated anti-CD11b, APC-conjugated anti-DX5, APC-conjugated anti-GR-1 (BD Biosciences, San Jose, CA) and PE-conjugated anti-murine LAIR-1 antibodies, (Affymetrix/eBioscience, San Diego, Ca). All were used according to the manufacturer’s recommendations.
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3

Antigen-Specific T Cell Activation Assay

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Expanded cells were restimulated for a period of 5–6 hours with either CMVpp65 Peptivator loaded autologous PBMCs or untouched autologous PBMCs as a control, all labelled with 1 µM CFSE (Sigma-Aldrich) at a ratio of 5∶1 at a concentration of 1×107/ml in 48 well plates. For analysis of intracellular cytokines, cells were incubated in the presence of anti-CD28 antibody (BD Bioscience) and 1 µg/ml of Brefeldin A (Sigma-Aldrich) added after 2 hours. Cells were fixed and permeabilized using Intrastain (DakoCytomation, Ely, UK) according to the manufacturer’s instructions and stained with CD25-APC, CD4-PerCP either PE-conjugated anti-IL-2, anti-TNF-α, anti-Granzyme B, anti-IL-10 or anti IFN-γ and CD69-APC Cy7 (all BD Biosciences) monoclonal antibodies. For surface staining cells were stained for 15 minutes with CD4-FITC, CD25-PE, CD8-PerCP, CD3-APC and CD69-APC Cy7 (all BD Biosciences) monoclonal antibodies.
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