Two-chain HK (HKa) and its LC can disaggregate LPS. (A) BODIPY FL–LPS at 50 ng/ml was suspended in 100 µl PBS. After the addition of BSA, HK, HKa, HC, or LC at 1 µg/ml, the real-time change in BODIPY FL–LPS fluorescence upon transition from the aggregated LPS state was recorded in 20-s intervals. SDS (2%) was used as the positive control. (B) BODIPY FL–LPS at 50 ng/ml was suspended in 100 µl PBS. After the addition of LBP, CD14, and LBP+CD14, or HKa at 1 µg/ml to each, the real-time change in BODIPY FL-LPS fluorescence was recorded in 20-s intervals. (C–G) BODIPY FL–LPS was suspended in 100 µl PBS at the indicated concentrations shown on the top of the graph. After 0.1, 0.5, or 2.5 µg/ml HKa was added, the real-time change in BODIPY FL–LPS fluorescence upon transition from the aggregated LPS state was recorded in 20-s intervals. The gain in fluorescence upon the addition of 2% SDS was used to define the full disaggregated state of BODIPY FL–LPS at 600 s (100% disaggregated); on this basis, the relative fluorescence intensity (RFI; %) of each curve was calculated, and the data are shown in H. Data are representative of three independent experiments and expressed as mean ± SEM.