Pacific blue anti mhcii

Bone marrow cells from C57BL/6 and P2X7R^−/− mice were cultured in BMDC culture media containing RPMI-1640 (Sigma-Aldrich) with 10% FBS (Thermo Fisher Scientific), 1% penicillin/streptomycin, 1% l-glutamine, 50 μM β-mercaptoethanol and 4% granulocyte–macrophage colony-stimulating factor. After 6 days of culture, the phenotype of BMDCs was confirmed by staining with PE-Cy7-anti-CD45, Alexa Fluor 700-anti-CD11c (eBioscience) and Pacific Blue-anti-MHCII (BioLegend) antibodies, and the purity of MHCII⁺CD45⁺CD11c⁺ BMDCs was >90%. Splenocytes (10⁶ cells per ml) from C57BL/6 OT-II mice were cocultured with OVA-pulsed BMDCs from WT or P2X7R^−/− mice at a ratio of 10:1 in a 48-well plate (BD Falcon) for 72 h.

Cells were incubated with an Fc receptor blocker, anti-CD16/32 antibody (2 μg ml⁻¹; eBioscience, Cheshire, UK). After washing, cells were incubated with FITC-anti-CD45 (eBioscience), Pacific Blue-anti-MHCII (BioLegend, London, UK), APC-anti-F4/80 (eBioscience), Alex Fluor 700-anti-CD11c (eBioscience), APC-Cy7-anti-11b (BD) and PerCP/Cy5.5-anti-CD103 (BioLegend) antibodies. For assessing intracellular cytokine expression, cells were labelled with FITC-anti-CD3 (BD) and PerCP-anti-CD4 (BD) antibodies, fixed and permeabilised using FOXP3 Fix/Perm buffer set (BioLegend) followed by staining with PE/Cy7-anti-CD25, Alexa Fluor 647-anti-FOXP3, BV-421-anti-IFN-γ, PE-IL-4 and BV-605 anti-IL-17 antibodies (all from BioLegend). Cells were acquired by flow cytometry on the BD LSRII (BD, Oxford, UK) and the data were analysed using FlowJo flow cytometry software (Tree Star, Ashland, OR, USA). The expression of P2X7R in CMT-93 cells was assessed by intracellular staining of P2X7R using FITC-conjugated rat anti-mouse P2X7R antibody (Aviva System Biology, San Diego, CA, USA) and FITC-conjugated rat IgG1 (BD) was used as isotype control.

Cells at (1 × 10⁶/ml) were collected and re-suspended in 300 μl FACS buffer. The cell suspension was then stained for flow cytometry analysis. Fc receptors were blocked using an anti-CD16/32 antibody (2 μg/ml), (eBioscience, Cheshire, UK). The cells were then washed and stained with FITC-anti-CD45 (1 μg/ml), AlexaFluor700-anti CD11c (1 μg/ml), APC-anti-F4/80(1 μl/ml), PE-antiCD86, (2 μg/ml), (all eBioscience), and pacific blue-anti-MHCII (1 μg/ml, BioLegend, London, UK), APC-cy7-anti-CD11b (1 μg/ml, BD, Plymouth, UK), PercP/Cy5.5-anti-CD103 (2 μg/ml) antibodies, and V510 (viability stain, 1 μg/ml).
Single stain controls were prepared using beads or spleen cells, negative controls were prepared using unstained cells. Cells were acquired by flow cytometry on the BD LSRII (BD) and the data were analyzed and visualized using FlowJo™ flow cytometry software (Ashland, OR: Becton, Dickinson and Company; 2019, Berkshire, UK; https://www.flowjo.com/).