Glass bottomed 384 well imaging plates

To select B cells producing mAbs, 10⁶ cells/mL-PBS were stained with 1 µM ER-Tracker™ Green (BODIPY^® FL Glibenclamide, Life Technologies, Foster City, CA) at room temperature for 5 min then centrifuged at 1,000 × g for 2 min as described previously^{6 (link)}. The cells with higher fluorescence intensity were then sorted by FACS (JSAN; Bay Bioscience, Kobe, Japan). Next, the sorted cells were selected by antigen-coated magnetic beads as described previously^{17 (link)}. In brief, biotinylated inactivated bacterial cells conjugated with DynaBeads M-280 Streptavidin (SA beads, Life Technologies) were prepared and these conjugates were used to concentrate B cells that bound to the antigens after removing cells non-specifically bound to SA-beads and E. coli DH5α-coated beads. After that, the remaining B cells were separated into each well (1 cell/10 µL-PBS) of 0.2 mm glass-bottomed 384-well imaging plates (Corning, Corning, NY, USA) and the bead-B cell complexes in each well were confirmed under a phase-contrast inverted microscope (CKX53; Olympus, Tokyo, Japan). Bürker-Türk plates were used for cell counting.

After a few mL of blood samples were collected from the immunized rabbits, lymphocytes were separated by a previously reported protocol using density-gradient centrifugation [11 (link)]. To select matured B cells, 10⁶ cells/mL-PBS were stained with 1 µM ER-Tracker™ Green (BODIPY^® FL Glibenclamide, Life Technologies, Foster City, CA, USA) at room temperature for 5 min then centrifuged at 1000× g for 2 min as described by Kurosawa et al. [2 (link)]. The cells with higher fluorescence intensity were then sorted by fluorescence activated cell sorting (FACS, JSAN; Bay Bioscience, Kobe, Japan). Next, the sorted cells were selected with antigen-coated magnetic beads as described previously [11 (link)]. In brief, biotinylated dead bacterial cells conjugated with DynaBeads M-280 Streptavidin (SA beads, Life Technologies) were prepared and these beads were used to concentrate B cells that bound to the antigens after removing cells non-specifically bound to SA-beads and E. coli DH5alpha-coated beads. After that, the remaining B cells were separated into each well (1 cell/10 µL-PBS) of 0.2 mm glass-bottomed 384-well imaging plates (Corning, Corning, NY, USA) and the bead-B cell complexes in each well were confirmed under a phase-contrast inverted microscope (CKX53; Olympus, Tokyo, Japan). Bürker-Türk plates were used for cell counting.