Celltrace yellow cell proliferation kit

Purified naïve B cells (2 × 10⁵ cells per well) were cultured in a 24-well tissue culture plate (Corning, Corning, NY, USA) in RPMI medium supplemented with 10% FBS (Sigma), 1 mM sodium pyruvate, 10 mM HEPES, 5.5 × 10⁻⁵ M β-ME, 100 units mL⁻¹ penicillin, and 100 μg mL⁻¹ streptomycin (Gibco, Waltham, MA, USA). Murine recombinant IL-4 (20 ng mL⁻¹, PeptroTech, Rocky Hill, NJ, USA) and BAFF (50 ng mL⁻¹, R&D Systems, Minneapolis, MN, USA) were added in the initial culture medium. For fluorescent proliferation analyses, isolated B cells were immediately stained with CellTrace Yellow Cell Proliferation Kit (Invitrogen) according to the manufacturer’s instructions prior to culture. On day 3 of culture, IL-4 was replaced with recombinant IL-21 (10 ng mL⁻¹, Peptrotech). CD40-CD40L ligation was provided using microbead-bound CD40L (100 ng mL⁻¹), as previously reported. In brief, superparamagnetic microbeads (diameter of 1 μm) with covalently attached anti-HA peptide antibody (Pierce, Waltham, MA, USA) were washed by repeating magnetic precipitation and resuspension in fresh PBS at least 3 times, before incubation with recombinant murine CD40L with N-terminal HA tag (R&D Systems) for 2 h at 4 °C under rotation. Anti-IgM antigen conditions were varied as specified in the text. Both anti-IgM and CD40L-microbeads were added to the initial culture on day 0 and supplemented on day 3.