Nitrocellulose bottomed 96 well plates

IFNγ ELISPOT was performed using antihuman IFNγ mAb1-D1K (I) and mAb7-B6–1 (II) (Mabtech) per the manufacturer’s protocol. Then 15,000 TEG cells (TEG011, TEGLM1, TEG011_CD8α, or TEGLM1_CD8α) were co-incubated with 50,000 target cells (E:T ratio 1:3) for 18–24 h in nitrocellulose-bottomed 96-well plates (Millipore). IFNγ spots were visualized with TMB substrate (Sanquin), and subsequently the number of spots was quantified using ELISPOT Analysis Software (Aelvis). Where indicated, blocking of CD8α was performed using 10 μg/ml anti-CD8α antibody clone OKT8 (eBioscience) and blocking of CD8β with 10 μg/ml anti-CD8β clone 2ST8.5H7 (Abcam).

Splenocytes were diluted to concentrations of 1 × 10⁵ and 1 × 10⁴ per ml in 96-well flat bottom plates (Nunc, Nalgene International, Rochester, NY). Plates were supplemented with 8 × 10⁵ irradiated (1,200 rads) syngeneic spleen cell feeders per well derived from naive mice.^{16 (link)} Concanavalin A (ConA)–supplemented master media mix (20 ng/mL PMA, 2.5 μg/mL ConA, 1 mg/mL R595 LPS, Sigma) was added to the stimulated wells and RPMI alone to the unstimulated wells, totaling to 200 µL volume per well.^{16 (link)} Cells were cultured for 6 days at 37°C, 5% CO₂, and 100% humidity. After culturing, each cell suspension was transferred to 96-well U-bottom plates (Nunc, Nalgene International) and centrifuged at 1,000 rpm in a tabletop centrifuge (Fisher Scientific). Cells were washed with 200 μL of RPMI complete media. Each cell pellet was resuspended in 100 μL of RPMI complete media and transferred to nitrocellulose-bottomed 96-well plates (Millipore) previously coated with anti-mouse IgG (Southern Biotech), Vi, CT, or KLH and processed as described earlier for the ASC assay. We excluded any samples that had spots in the unstimulated well for antigen-specific responses, or more than two spots in the KLH well after stimulation. We used stimulation of approximately 150% over unstimulated wells for the total number of memory B cells to consider the results to be usable for analysis.