Human B cells (preenriched, enriched, and depleted) from six donors were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Gibco, Fisher Scientific) supplemented with 10% HI FBS (Gibco), 2:1,000 MycoZap (Lonza, Alpharetta, GA), 10,000 U/ml interleukin-2 (IL-2; Gibco), and 100 μg/ml IL-21 (BioLegend). Additionally, the medium was treated with antibody-cross-linked CD40L (Miltenyi Biotec), according to the manufacturer’s protocols, or 1 ng/ml CD40L homotrimer (BioLegend) in plates coated with 10 μg/ml anti-His antibody (BioLegend) or 3T3-msCD40L feeder cells obtained through the NIH AIDS Reagent Program (catalog no. 12535 3T3-msCD40L; Division of AIDS, NIAID, NIH, from Mark Connors, as described in reference 22 (link)). Although three different CD40L delivery methods were used, no statistical difference in IgGtot production was found between the three (not shown). B cells were plated with 3,000 to 5,000 cells/ml and incubated at 37°C in 5% CO2 for up to 12 days, after which the supernatant was collected and stored at –80°C for downstream analysis.
Lamb C.L., Price E., Field K.P., Dayton C., McIndoo E.R., Katahira E.J., Stevens D.L, & Hobdey S.E. (2019). Enrichment of Antigen-Specific Class-Switched B Cells from Individuals Naturally Immunized by Infection with Group A Streptococcus. mSphere, 4(6), e00598-19.
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