Naïve B Cell Isolation and Differentiation

Purified naïve splenic B cells were isolated from mice by first using a percoll (GE Healthcare, IL, USA) gradient (80/65/50% in PBS, cells collected from 80/65% interface), followed by negative isolation with a B cell isolation kit (Miltenyi Biotec), as described previously [9 (link), 12 (link), 40 (link)]. Cells were labeled with division tracking dye, Cell Trace Violet (CTV). Purity of B cell population was verified as >95% B220⁺ CD19⁺ by flow cytometry. Labeled B cells were stimulated with lipopolysaccharide (LPS) derived from Escherichia coli 026:B6 (15 µg/mL; Sigma). For in vitro differentiation studies, B cells were stained using antibodies to CD138 (clone 281-2, BD Pharmingen). Triplicate plates were set up on the first day and left undisturbed – one plate was analyzed at each timepoint. All lymphocytes were incubated at 37 °C with 5% CO₂ and humidity control.