Splenocytes were prepared for flow cytometric analysis as previously described [32 (link)]. Splenocytes were stimulated with Tg ME49 Ag (4 μg/mL) for 2 hr at 37 °C with 5% CO2. After antigen stimulation, cells were stained with fluorophore-conjugated GL7 (PE) and B220 (FITC) antibodies purchased from BD Biosciences (Franklin Lakes, NJ, USA) and Invitrogen (Waltham, MA, USA). All flow cytometry procedures were performed according to the manufacturer’s protocol. Stained samples were analyzed using the Accuri C6 flow cytometer and the C6 Accuri software (BD Biosciences, Franklin Lakes, NJ, USA).
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