Fl600 fluorescent plate reader

The change in the mitochondrial membrane potential (ΔΨm) in HLE-B3 cells was monitored using the mitochondrial membrane potential detection kit (JC-1, T4069, Sigma-Aldrich) according to the manufacturer's instructions. Briefly, HLE-B3 cells cultured in a 96-well plate (5 × 10³ cells per well) were treated with Tat followed by treatment with 1x JC-1 reagent diluted in serum-free DMEM for 20 min at 37°C in a 5% CO₂ atmosphere. Thereafter, cells were rinsed once with 1x rinsing buffer provided with the kit. Fluorescence was measured using the FL600 fluorescent plate reader (BioTek Instruments, Winooski, VT, USA) at excitation wavelengths of 485 and 535 nm. All experiments were repeated at least three times.

The activities of caspase-3 and caspase-9 were evaluated using Caspase-3 Fluorescent Assay Kits and Caspase-9 Fluorescence Assay Kits (Clontech, Palo Alto, CA), respectively, according to the company's specifications. Briefly, cells under various conditions were lysed with lysis buffer and then centrifuged at 4°C to prepare the supernatants. Then, the reaction buffer containing 5 μl of the specific substrate of DEVD-AFC for caspase-3 or LEHD-AMC for caspase-9 was supplemented into supernatants and incubated for 1 h at 37°C. All specimens were subsequently analyzed using an FL600 fluorescent plate reader (BioTek Instruments, VT, USA) to determine the fluorescent intensity.