Fig. 6.

Bupivacaine induces permeabilization of the lysosomal membrane. (A) Histogram representing the statistical analysis shows the MFI of green fluorescence in the AO relocation experiment. IVD cells were first preincubated with 5 μg/ml AO solution for 15 min with complete medium and then exposed to bupivacaine for 60 min. Green fluorescence was determined by flow cytometry. Data are presented as the means ± SD of three independent experiments (^*P < 0.05 vs. saline control). (B) Representative graphs of the AO uptake experiment obtained by flow cytometry analysis. IVD cells were first treated with bupivacaine for 60 min and then stained with 5 μg/ml AO solution in complete medium. Cells deficient in intact lysosomes (“pale cells”) were determined by flow cytometry. (C) Histogram analysis showing the percentage of “pale cells”. Data are presented as the means ± SD of three independent experiments (^*P < 0.05 vs. saline control). (D) Typical confocal images of in situ AO staining (Magnification ×400, scale bars = 50 µm). AF, annulus fibrosus; NP, nucleus pulposus; B, bupivacaine.