Ctl immunospot s6 ultimate 5 analyzer

Cryopreserved PBMC were thawed and placed in RPMI 1640 medium supplemented with 10% heat-inactivated normal human serum (100–318, Gemini Bio-Products), L-glutamine, penicillin, and streptomycin. After an overnight rest at 37 °C, the PBMC were washed, resuspended in serum-free medium (SFM; X-VIVO 15, Lonza), and 1–2 × 10⁵ cells were plated per well of a 96-well Millipore MAIPSWU plate coated with anti-IFNγ antibody according to the manufacturer’s instructions (3420-2HW-Plus, Mabtech Inc.). Peptide pools were added to the cells at a final concentration of 1μg/mL/peptide prior to incubation at 37 °C overnight. Controls included SFM plus 0.5% DMSO (negative) and anti-CD3 (positive). The ELISpot plates were developed using TMB substrate and read using a CTL-ImmunoSpot® S6 Ultimate-V Analyzer (Cellular Technology Limited). For the T cell epitope mapping studies using matrixed peptide pools, positive wells were defined as those with SFC/10⁶ PBMC values five-fold over background (negative/no stimulation control) and greater than 5 SFC/10⁶ PBMC after subtraction of the negative control.

Memory B cell ELISpots were performed using the Human IgG ELISpot Basic kit (catalog no. 3850-2H, MabTech). Cryopreserved PBMC were thawed and placed into culture for 6 days with R848 plus IL-2 in R10 medium (RPMI 1640, 10% FBS, penicillin/streptomycin, l-glutamine) to stimulate the production of antibody by memory B cells. MAIPSWU10 plates were coated with PBS (negative control), anti-IgG antibody (positive control), live virus (DENV-1 strain WestPac74, DENV-2 S16803, DENV-3 CH53489, and DENV-4 TVP-360), or 15 µg/mL recombinant E (rE) protein from DENV-1–4 (Hawaii Biotech) and incubated overnight at 4 °C. After washing off the unbound virus, 100,000–200,000 harvested memory B cells were plated into each well of the plate. Plates were incubated at 37 °C overnight, after which cells were removed and spots were developed using horseradish peroxidase-conjugated anti-IgG secondary antibody and TMB substrate. Spots were counted using a CTL ImmunoSpot S6 Ultimate-V Analyzer (Cellular Technology Limited) and counts reported as spot-forming units per million input cells.