Capture anti mouse ifn γ mab

Capture anti-mouse IFN-γ mAb (clone: R4–6A2) and biotinylated, detection anti-mouse IFN-γ mAb (clone: XMG1.2-Biotin) were purchased from BioLegend, San Diego, CA. Peroxidase-conjugated AffiniPure Donkey Anti-Mouse lgM and Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) were purchased from Jackson ImmunoResearch Inc. Anti-mouse CD-3 antibody (clone: 145–2C11) was purchased from BioXCell, West Lebanon, NH. FITC-, PE-, APC- or biotin-conjugated monoclonal antibodies to Foxp3 (clone: MF-14), B220 (clone: RA3–6B2), CD4 (clone: GK1.5), and CD8 (clone: 53–6.7) were purchased from BioLegend, San Diego, CA.

The assay method was described previously [22 (link)]. Briefly, splenocytes collected from immunized mice were reactivated by pOVA (2.5 mg/mL) for 48 h before being loaded into wells of 96-well filtration plates (Millipore, Billerica, MA, USA). The wells were precoated with 5 mg/mL of capture anti-mouse IFN-γ mAb (Clone: R4–6A2, Biolegend, San Diego, CA, USA). Triplicates were set up for each condition. Cells were discarded after 24 h, and the wells were incubated overnight with 2 mg/mL of biotinylated detection anti-mouse IFN-γ mAb (Clone: XMG1.2-Biotin, Biolegend, San Diego, CA, USA). After washing, the bound anti-mouse IFN-γ mAb was detected using horseradish peroxidase (HRP Avidin, Biolegend, San Diego, CA, USA) together with a 3-amino-9-ethyl-carbazole (AEC) substrate (Sigma, St. Louis, MO, USA). The bottoms of the wells were scanned, and the spots on the bottom of the well were automatically counted using ImageJ software.