24 well flat bottom culture plates

Cells were maintained in 24-well flat-bottom culture plates (Corning) and IMDM supplemented with Glutamax and 10% heat-inactivated FBS (Invitrogen), Hybridomax hybridoma growth supplement (11 μl/ml), lipid mixture 1, chemically defined, and MEM amino acids solution (both at 1× final concentration) from day 3 onwards.

24-well flat-bottom culture plates (Corning) and IMDM supplemented with GlutaMAX and 10% heat-inactivated FBS (HIFBS; Invitrogen) were used. Day 0 to day 3: B-cells were cultured at 2.5 × 10⁵/ml with IL-2 (20 U/ml), IL-21 (50 ng/ml), and F(ab′)₂ goat antihuman IgM & IgG (10 μg/ml) on γ-irradiated CD40L expressing L cells (6.25 × 10⁴/well). Day 3 to day 6: at day 3, the cells were detached from the CD40L L-cell layer and reseeded at 1 × 10⁵/ml in media supplemented with IL-2 (20 U/ml), IL-21 (50 ng/ml), Hybridomax hybridoma growth supplement (11 μl/ml), lipid mixture 1, chemically defined and MEM Amino Acids Solution (both at 1× final concentration). For UNC0638 experiments, ABCs were treated at day 3 with inhibitor at the indicated concentration (generally 2 μM), vehicle control (DMSO), or with standard conditions as indicated. The cells were sampled at the indicated time points without further addition of inhibitor. NCI-H929 and U266 cells (DSMZ) were cultured in RPMI1640 media and OCI-LY3 and OCI-LY10 (Prof R. E. Davis lab) in IMDM with GlutaMAX (Life Technologies), each containing 10% heat inactivated fetal calf serum (51 (link)). Cell lines identity was confirmed using short tandem repeat profiling.