Isolation and Activation of Human and Murine B Cells

Human peripheral blood mononuclear cells were donated by the Chungbuk Red Cross Blood Center (Cheongju, Korea). Lymphocytes were isolated from these cells by density gradient centrifugation (Ficoll-Paque) 34 (link). hB cells were isolated from lymphocytes using a human B cell isolation kit (Miltenyi Biotec, Auburn, CA, USA). mB cells were purified from spleen cells of MRL.Fas^lpr mice by a negative depletion method using mouse B cell isolation kit (Miltenyi Biotec) 9 (link). Purity of hB and mB cells was typically >90%. B cells (1 × 10⁵ cells/well) and MSCs (0.01-0.1 × 10⁵ cells/well) were added in 200 µL to the wells of 96-well plates. CpG-oligodeoxynucleotide (ODN; 5 μg/mL) was used to activate hB cells, and lipopolysaccharide (LPS; 1 μg/mL) was used to activate mB cells. To measure B cell proliferation, cells were pulsed with [³H]-thymidine (113 Ci/nmol; NEN, Boston, MA, USA) at a concentration of 1 μCi/well for the last 18 h and were harvested on day 3 using an automated cell harvester (Inotech, Dottikon, Switzerland). The amount of [³H]-thymidine incorporated into the cells was measured using a Wallac Microbeta scintillation counter (Wallac, Turku, Finland) 3 (link).