Alexa fluor 647 anti foxp3

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.

The obtained blood samples were separated to plasma and lymphocytes. Lymphocytes were stained (surface and intracellular) to determine Treg⁺ cells CD4⁺CD25⁺FOXP3⁺ and Th1 CD3⁺CD4⁺CD25⁻ population in treated groups in comparison to PBS. First, cells were stained by Percp-Anti-CD3, FITC-Anti-CD4, and PE-Anti-CD25 (Biolegend, USA) for 30 min on ice. Then it was fixed, permeabilized and stained by Alexa Fluor 647- anti- Foxp3 (Biolegend, USA) for 30 min on ice. Finally cells were examined by BD Accuri C6. Later, plasma samples were utilized to determine periphery TGF-β and IL-12 cytokines by ELISA kits according to manufacturer instructions (Lington Biosciences, China).