Mhcii apc efluor 780

HDM was from Greer Laboratories. Anti-mouse IL-1β (p17) (AF-401-NA) was from R&D. Anti-mouse caspase-1 (p20) (AG-20B-0042) and anti-NLRP3 (AG-20B-0014) antibodies were from Adipogen. Anti-ASC (67824) antibody was from Cell Signaling Technology. Anti-β-actin (66009-1-Ig) was from Proteintech.
Anti-mouse antibodies used for flow cytometry were: CD3-FITC (BD, 553062, 145-2C11), CD19-FITC (eBioscience, 11-0193-82, 1D3), Ly-6G-PE (Biolegend, 127608, 1A8), CD45-PE (eBioscience, 12-0451-81, 30-F11), CD11c-PerCP-Cy5.5 (Biolegend, 117328, N418), CD11b-PerCP-Cy5.5 (BD, 550993, M1/70), CD11b-PE-Cy7 (eBioscience, 25-0112-82, M1/70), CD3e-PE-Cy7 (BD, 552774, 145-2C11), CD19-PE-Cy7 (Biolegend, 115520, 6D5), SiglecF-Alexa Fluor 647 (BD, 562680, E50-2440), MHCII-APC-eFluor 780 (eBioscience, 47-5321-82, M5/114.15.2), Ly-6G-BV421 (Biolegend, 127628, 1A8), CD45-BV510 (Biolegend, 103137, 30-F11), CD11c-BV510 (Biolegend, 117338, N418). Ultrapure LPS was obtained from Invitrogen. Nigericin was obtained from Sigma-Aldrich. RRx-001 (S8405) was from Selleck.

Heparinized whole blood was used for flow cytometry using the following antibody cocktail: CD45-PerCP (Clone 30-F11; Biolegend 103130), CD3-eFLUO450 (Clone 17A2; eBioscience 48-0032), NK1.1-PE (Clone PK136; BD557391), Ly6G-APC-CY7 (Clone 1A8; BD560600), CD11b PE-CY7 (Clone M1/70; BD552850), Ly6C-APC (Clone 1G7.G10; Miltenyi 130-093-136), CD19-APC-H7 (Clone 1D3, eBioscience 47-0193) and Siglec-F-PE (Clone E50-2440; BD552126). Further, cells from the whole brain were isolated for FACS. Anesthetized mice were perfused with PBS to remove the peripheral blood. Subsequently, brains were dissected, mechanically dissociated and digested with a collagenase mix (including collagenase IX, collagenase I, DNAse and RPMI-HEPES). Immune cells were separated using a Percoll-gradient and stained with CD45 PerCP (Clone 30-F11; Biolegend 103130), CD11c PE-Cy7 (Clone N418, eBioscience 25-0114)), F4/80 (Clone BM8; Biolegend 123116), CD11b BV510 (Clone M1/70; Biolegend 101245), CD3 BV421 (Clone 17A2; eBioscience 48-0032-82), CD19 BV421 (Clone 6D5; Biolegend 115538), Ly6G BV421 (Clone eBio927; eBioscience 48-3172), MHC II-APC-eFluor780 (Clone M5/114.15.2; eBioscience 47-5321-82). All samples were measured with a FACS-Canto II (BD Biosciences). Results were analysed with FACSdiva version 8 (BD Biosciences).

Flow cytometry was used to determine the number and types of cells present in the BAL fluid. Fc-blocked (1 µg/ml; eBiosciences) BAL cells were stained with anti-mouse SiglecF-phycoerythrin (PE; 1 µg/ml; BD Pharmingen), CD45- allophycocyanin (APC; 1 µg/ml; eBiosciences), CD3-PECy5 (1 µg/ml; eBiosciences), CD19-PECy5 (1 µg/ml; eBiosciences), CD11c-PECy7 (1 µg/ml; eBiosciences), MHCII-APC-efluor780 (1 µg/ml; eBiosciences), CD11b-V450 (1 µg/ml; BD Pharmingen), Ly6C-FITC 1 µg/ml; BD Pharmingen) and Ly6G-AlexaFluor700 (1 µg/ml; BD Pharmingen). Fixable Viability Dye eFluor^® 506 (eBiosciences) was added to exclude dead cells from the analysis.