Association between HMGB1 release and NLC differentiation. (A) Association between cell viability and NLC differentiation. Cell viability of each sample was monitored every 2 or 3 days using a Cell Viability Analyzer (Beckman Coulter). The box represents the day on which NLCs were first identified. (B) Observation of NLCs by MTT staining. After 1 or 2 weeks of culture, the cells in suspension were removed. Cells were then cultured with fresh medium containing 0.5 mg/mL MTT for 2 hours. (C) HMGB1 passive release. CLL cells were cultured for 2 days on chambered slides. After fix/permeabilization, cells were costained with rabbit anti-COX IV (red) and mouse anti-HMGB1 (green) antibodies. Arrows indicate a typical phenomenon of HMGB1 passive release from the cells. (D-E) Time course of HMGB1 release determined by (D) ELISA and (E) western blotting in 2 representative cases of 6 independent cases examined. HMGB1 release into culture medium was monitored over 19 days. Conditioned medium was collected every 2 or 3 days and stored at −80°C. Ten or 40 µL of conditioned medium was used for ELISA assay or western blotting, respectively. Western blotting was probed with an anti-HMGB1 mouse antibody first and then reprobed with an anti-LDH rabbit antibody. (G) Determination of HMGB1 release by western blotting. Four CLL samples were cultured for 15 days, and conditioned medium was collected in every 5 days. Numbers of NLCs were quantified by MTT staining at the 15th day. The amounts of NLCs indicated as −, +, ++, +++, and ++++ were 0%, 10%, 25%, 50%, and >75% coverage of the well. (F,H) DNA concentration in the conditioned medium. Two hundred microliters of conditioned medium was used to determine DNA concentration. Boxes in A, D, E, and F indicate the time when NLCs were observed. Numbers in each chart are CLL sample IDs.