Horizon violet cell proliferation dye 450

B cell purification was performed with the EasySep Mouse B Cell Enrichment Kit (StemCell Technologies). 1×10⁷ cells/ml were incubated in PBS with 0.5% (v/v) fetal calf serum (FCS, HyClone) and 4µM of BDHorizon Violet Cell Proliferation Dye 450 (VPD450, BD Biosciences) for 5min at 37°C and the reaction was quenched with 3 volumes of FCS for 1min at 37°C and cells were washed twice in RPMI 1640 medium. Cells were cultured in the presence of 5µg/ml CpG ODN 1826 (5’-tccatgacgttcctgacgtt-3’; Invivogen) for 3–4d at 37°C and then subjected to flow cytometric staining.
Vλ1 sequencing was performed essentially as described (Anderson et al., 2007 (link)).
Immunohistological analysis was performed essentially as described (Kerfoot et al., 2011 (link)). EdU was detected using the Click-iT^® EdU Alexa Fluor 555 Imaging Kit (Invitrogen). 40x tiled images of whole splenic sections were acquired with an IX83 fluorescent microscope (Olympus) and image analysis was performed using cellSens Dimension software (Olympus) with the count and measure module.