Biotek cytation reader 3

Mitochondrial apoptotic cascade involves the depolarization of mitochondrial membrane, which can be measured fluorometrically using JC1 dye (JC-1; CAS 47729-63-5; Calbiochem; Sigma-Aldrich, USA). Briefly, 5 × 10⁴ cells were seeded per well in 96-well plates and treated as described in Section 2. After the treatment, cells were incubated with JC1 staining solution (5 µg/mL) at 37 °C for 20 min followed by washing of the cells twice with PBS. Mitochondrial membrane potential was estimated by measuring the fluorescence ratio of JC1 aggregates in mitochondria (red) (λ_ex 550 nm, λ_em 600 nm) to that of free JC1 monomers (green) (λ_ex 485 nm and λ_em 535 nm) using a fluorescence plate reader (Biotek Cytation Reader 3; BioTek Instruments, Inc., Winooski, VT, USA). Mitochondrial membrane depolarization is indicated by an increase in the proportion of green JC1 monomers in mitochondria, whereas repolarization of healthy mitochondria is indicated by an increase in red JC1 aggregates.

ROS production was assayed using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) from a Sigma-Aldrich (St. Louis, MO, USA) assay-specific probe. Immediately after the menadione and picein treatment, the medium was removed from the 48-well plates and replaced with the assay-specific probe DCF-DA (40 μM) in 0.5 mL of Hank’s balanced salt solution and thereafter incubated for 30 min in the incubator. Further, fluorescence was measured at 485 nm excitation/535 nm emission wavelengths by a Biotek Cytation Reader 3 (BioTek Instruments, Inc., Winooski, VT, USA).

Cells were seeded in 96-well plate and then subjected to the different treatments. After incubation time, the sulforhodamine B (SRB) assay was performed for cell mass determination based on the measurement of cellular protein content [31 (link)]. Briefly, the cell culture medium was discarded, and wells rinsed with PBS 1X. Cells were fixed by adding 1% acetic acid in 100% methanol for at least 2 h at −20 °C. The fixation solution was then discarded, and the plates were dried at 37 °C. 150 µL of 0.05% SRB in 1% acetic acid solution was added and incubated at 37 °C for 1 h. The wells were then washed with 1% acetic acid in water and dried. Then, 100 μL of Tris (pH 10) was added and the plates were stirred for 15 min and optical density was measured at 540 nm in Biotek Cytation 3 reader (Biotek Instruments, Winooski, VT, USA).