Miltenyi pan b cell isolation kit 2

Aldehyde sulfate beads were prepared at a concentration of 1.0 x 10⁶ beads/100 μL ice-cold blocking buffer, aliquoted to a standard 48 well plate, and imaged by brightfield microscopy using a Lionheart FX automated live cell imager (Biotek). To stain beads directly for fluorescent imaging, 4 μL beads were combined with 4 μg donkey anti-rat IgG AF594 (Life Technologies) in 50 μL PBS, followed by a 40 min incubation at room temperature. The sample was quenched with ice-cold blocking buffer, washed three times, and resuspended at a concentration of 1.0 x 10⁶ beads/100 μL ice-cold blocking buffer for imaging. B cells were isolated from C57BL/6 whole spleen single cell suspensions using the Miltenyi Pan B Cell Isolation Kit II (Miltenyi) and prepared at 1.0 x 10⁶ beads/100 μL ice-cold blocking buffer. Fluorescent beads and B cells were then mixed at a ratio of 50 μL:50 μL in a 48 well plate, incubated for 40 min, and imaged.

Whole spleens from C57BL/6 mice were first homogenized by gentle mechanical disruption with a syringe plunger and 70 μm mesh filter. B cells were subsequently isolated using the Miltenyi Pan B Cell Isolation Kit II (Miltenyi), in accordance to the manufacturer’s instructions.