Pe cy7 anti ifnγ

PerCP-Cy5.5 conjugated antibody to CD3 (cat 45–0031-82, clone 145-2C11); APC or APC-eFluor780-anti-CD8 (cat 17–0081-82, 47–0081-82, clone 53–6.7); eFluor450-anti-CD279 (PD-1) antibody (cat 48–9985-82, clone J43), PE-Cy7 anti-IFNγ (cat 25–7311-82, clone XMG1.2); PE-anti-Ki-67 (cat 12–5698-82, clone SolA15), APC-eFluor780 labelled antibody to CD44 (cat 47–0441-82, clone IM7); and PE-Tim3 (cat 12–5870-82, clone RMT3-23) were obtained from eBioscience. PE-anti-mouse CD223 (LAG-3) antibody (cat 125,208 clone C9B7W), APC Annexin V (cat 640,920), and Propidium iodide (PI, cat 421,301) were from Biolegend. For analysis of IFNγ producing cells, the cells were stimulated with 50 ng/ml PMA and 200 ng/ml ionomycin in the presence of Golgistop (BD Biosciences) for 3 h and then fixed with the Foxp3 staining kit (eBioscience) and analysed by flow cytometry. For all flow cytometry analyses, data were acquired on a LSR II or Canto II (BD Immunocytometry Systems) and analysed using FlowJo (Tree Star).

Splenocytes harvested from C57BL/6, BALB/c mice were incubated with golgistop (2 µM of monensin, BioLegend, San Diego, CA, USA) for 2 h at 37 °C. Washed pellets were stained using the Zombie Violet Fixable Viability Kit (BioLegend, San Diego, CA, USA) at room temperature for 15 min to identify dead cells. Pellets stained with Zombie were washed and stained with surface antibodies for 20 min at 4 °C. After washing with PBS, the cells were fixed and permeabilized using Cytofix/Cytoperm buffer (eBioscience, San Diego, CA, USA) for 30 min. The cells were washed with Perm/Wash buffer (eBioscience, San Diego, CA, USA), and incubated with intracellular antibodies, PE-Cy7-anti-IFN-γ, PE-anti-perforin, and APC-anti-granzyme B at room temperature for 30 min. After washing with Perm/Wash buffer, the cells were resuspended in PBS and analyzed by flow cytometry. This experiment was performed after pre-staining the cells with unconjugated isotype control antibodies and Fc receptor binding antibodies to block the nonspecific binding of fluorescence-conjugated antibodies.

PerCP-Cy5.5-conjugated anti-TNFα, eFluor® 660 anti-GM-CSF, PE-Cy7 anti-IFNγ, PE anti-IL-17A, FITC-anti-CD45RA, PE-anti-CD45RO, eFluor® 450-anti-CD161 and unconjugated anti-CD28 antibodies (Abs) were from eBioscience. Brilliant Violet 605-anti-CCR6 and APC-anti-IL-10 Abs were purchased from BioLegend. RosetteSep negative-enrichment kits for human T cells, B cells and monocytes were from StemCell Technologies. Ficoll-Paque was from GE Healthcare, RPMI from Lonza and foetal bovine serum from Life Technologies. Lipopolysaccharide (E. coli, serotype 0111:B4), ionomycin and PMA were from Sigma-Aldrich and Golgiplug™ was from BD Bioscience and Leucoperm from AbD Serotec.