Tritiated 3h thymidine

Purified naïve B cells or T cells were plated at 1×10⁵ cells/well in triplicate (200 μl/well) in 96-well flat-bottom microtiter plates and stimulated as described above. Four days later, wells were pulsed with 1 μCi of tritiated (³H-) thymidine (Perkin Elmer) and incubated for 24 hrs prior to collection on a mash harvester. Incorporation of ³H-thymidine was quantified as counts per million. Alternatively, naïve B cells were labeled with PBS containing 0.5 μM carboxyfluorescin succinyl ester (CFSE, Invitrogen), washed 2x in complete RPMI, and stimulated as above for 5 days prior to flow cytometric analysis. In separate experiments, cell supernatants were harvested in duplicate on day 6 post-stimulation with the reagents described above. IgM and IgG levels were measured using Ready-Set-Go ELISA kits (eBioscience) according to the manufacturer’s instructions. For B cell survival assays, cell viability of resting or activated B cells was measured over 5 days using a flow cytometer, using forward/side scatter parameters and propidium iodide exclusion to gate on viable cells.