Vector blue substrate kit 3

EliSpot assays were performed as previously described [39 (link)]. Briefly, 96-well filter plates (Millipore) were coated with 2 ug/ml purified rat anti-mouse IL-2 (clone JES6-1A12, BD Biosciences) in PBS overnight at 4 °C, washed with media to remove unbound antibody and incubated with 100 ul media per well for 1 h to block non-specific binding. Isolated CD4 T cells were co-cultured with syngeneic spleen cells or transfected fibroblast antigen presenting cell (APC) (for HLA-DR1 transgenic mice [39 (link)]) and peptides for 16–20 h at 37 °C and 5% CO₂. The cells were removed from the plates, and the plates were washed with wash buffer (1X PBS, 0.1% Tween-20). Biotinylated rat anti-mouse IL-2 (clone JES6-5H4, BD Biosciences) was added at a concentration of 2 ug/ml, 50 ul/well, in wash buffer with 10% FBS (Fetal Bovine Serum) and incubated at room temperature for 30 min. The plates were washed again and streptavidin-conjugated alkaline phosphatase (Jackson Immuno Research) was added at a dilution of 1:1000 in wash buffer with 10% FBS, 50 ul/well, and incubated for 30 min at room temperature. The plates were and developed using Vector Blue substrate kit III (Vector Laboratories, City, CA, USA) prepared in 100 mM Tris, pH 8.2. After drying, quantification of spots was performed with an Immunospot reader series 5.2, using Immunospot software, version 5.1.

HA-specific ASCs were detected by B Cell ELISpot^{69 (link)}. Briefly, 96-well filter plates (Millipore, Billerica, MA, USA) were coated with 10 μg/mL purified A/New/Caledonia/99 HA in PBS overnight at 4 °C. Prior to plating, wells were washed with media to remove unbound HA, and incubated with media for 1 h at room temperature to block non-specific binding. Media was removed and cell suspensions were incubated for 4 h at 37 °C with 5% CO₂. Cells were subsequently removed from the filter plates and washed with ELISpot wash buffer (1X PBS with 0.1% Tween-20). Alkaline phosphatase-conjugated goat anti-mouse IgG (Southern Biotechnology#1030-04) diluted to 2 μg/mL in PBS containing 5% bovine serum albumin was added (100 μL/well), and the plates were incubated overnight at 4 °C. Plates were washed with ELISpot wash buffer and incubated with Vector Blue substrate kit III (Vector Laboratories, CA, USA) in 100 mM Tris (pH 8.2) for five minutes at room temperature. Following development, plates were washed with water and dried. Quantification of spots was performed using an Immunospot reader series 5.2 with Immunospot software version 5.1.