Phagocytosis by Trout B Cell Subsets

Phagocytosis by trout IgM⁺ and IgT⁺ B cells was performed as described previously (8 (link)). Briefly, fluorescent beads (Fluoresbrite Yellow Green Microspheres, 1.0 µm in diameter; Polysciences) in 300 μl L-15 medium (Sigma-Aldrich) were seeded in 24-well plates (Nunc) at a density of 10⁷ beads/well and pelleted by centrifugation at 2,500 g for 5 min. Then trout PBLs or HKLs in 300 μl L-15 medium were added to each well at a cell:bead ratio of 1:10, followed by incubation for 3 h at 17°C. After incubation, cell suspensions were centrifuged (100 g for 10 min at 4°C) over a cushion of 3% (weight/volume) BSA (Thermo Scientific) in PBS supplemented with 4.5% d-glucose (Sigma-Aldrich) to remove the non-ingested beads. The collected cells were stained with anti-trout IgM and anti-trout IgT mAbs as described above, followed by FACS to sort the phagocytic and non-phagocytic IgM⁺ and IgT⁺ B cells using BD FACSAria III (BD Biosciences). Cells were collected and subjected to total RNA isolation and cDNA synthesis as described above. The relative expression levels of AMP genes in the phagocytic and non-phagocytic trout B cells were determined by the Ct method and normalized against the internal control EF-1a using the 2^−ΔΔCt method (34 (link)).