Automax cell sorting machine

Splenocytes from primed old and young mice were sorted using magnet beads- labeled anti-mouse IgM and anti-mouse IgG (Miltenyi Biotec Inc. Auburn, CA) with AutoMax cell sorting machine (Miltenyi Biotec Inc. Auburn, CA) for the isolation of IgM⁺ cells and the depletion of IgG⁺ cells, respectively, by following manufacturer’s instruction. In brief, cells were blocked with Fc block antibodies at 4°C for 30 minutes, washed three times with running buffer (5% FBS and 0.2 mM EDTA in 1× PBS), and incubated with magnet- labeled antibodies (20 µl/10⁷ cells in 100 µl total volume) at 4°C for 30 minutes followed by three washes as before. Cells were sorted by cell sorting machine, and the purities of sorted cells, which were evaluated with flow cytometry, were equal to or greater than 98%. IgM⁺ cells, IgM⁻ cells, IgG⁺ cells, IgG⁻ cells and unsorted cells were cultured each with IL-4 (20 ng/ml) and the stimulator cells (splenocytes infected with influenza A/Taiwan virus for 4 hours and irradiated 3000 Rads of 6°C). Prior to and after three days of culture, different phenotypes of cells were respectively examined for IgG and IgM secreting cells with HA-specific ELISPOT assay, and HA-specific IgG and IgM in supernatant of cell culture on day 3 following the culture were assessed with ELISA as described above.