Carboxyfluorescein succinimidyl ester (cfse)

Mouse spleen B cells were prepared as described previously (Nomura et al., 1996 (link)). Cells were labeled with 2 µM CFSE (Molecular Probes) for 10 min. 2 × 10⁵ CFSE-labeled cells were cultured in 200 µl RPMI 1640 medium (Wako Pure Chemical Industries) supplemented with 10% FCS (Nichirei Biosciences), 50 µM 2-mercaptoethanol (Sigma), and 1% penicillin/streptomycin (Nacalai Tesque) in a 96-well plate with F(ab’)₂ fragments of goat anti–mouse IgM antibody (Jackson ImmunoResearch Laboratories, Inc.), NP-BSA, NP-Sm/RNP, imiquimod (InvivoGen), or LPS (Sigma-Aldrich). After 48 or 72 h, cells were analyzed by flow cytometry using FACS Verse flow cytometer (BD). For endocytosis assay, cells were incubated with 1 µg/ml NP-BSA or NP-Sm/RNP on ice for 30 min. After washing, 2 × 10⁵ cells in 100 µl complete culture medium were incubated at 37°C for various time. The reaction was terminated by addition of ice-cold PBS containing 2% FCS. Cells were stained with biotinylated anti-NP antibody (C6; a gift from Y. Takahashi, National Institute of Infectious Diseases, Tokyo, Japan) together with Alexa Fluor 647–conjugated streptavidin (Molecular Probes) and analyzed by FACS Verse flow cytometer (BD).