R595 lps

Single cell suspensions were prepared from spleen in complete IMDM medium (Gibco, Waltham, MA) with 10% FBS, 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 5×10⁻⁵ M of 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO). Lymphocytes were counted and diluted to 5×10⁶ cells/mL. 2.5×10⁶ cells/well were added into 48-well plates. For lipopolysaccharides (LPS) stimulation wells, cells were cultured with 0.4 μg R595 LPS (Sigma-Aldrich, St. Louis, MO), and 100 μL of supernatant from concanavalin A-stimulated splenocytes, prepared as previously described [30 (link)]. Influenza virus-specific in vitro stimulation was performed as previously described [31 (link)]. Briefly, mouse splenocytes were cultured for 6 days with 10 μg β-propiolactone (BPL)-inactivated A/Cal09 (IRR, FR1184), H3N2 control antigen 2009–2013 (FR43), and B/Brisbane/08 (B/Bris08, FR 1188), respectively. The culture supernatants were harvested and stored at -80°C for mPlex-Flu assay. For the mPlex-Flu assay, all samples were thawed, diluted and assayed at same time to minimize batch effects.

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.