Plate chameleontmv

T cells were seeded in 96-well plates, activated with anti-CD3/CD28-coated beads and treated for 1 or 5 days with PMT. Caspase 3, 7, and 8 activities were measured using the Caspase-3/7Glo and Caspase-8Glo Assay (Promega) in accordance to the manufacturer’s instructions. Luminescence was measured using the Plate CHAMELEON^TMV microplate reader (Hidex).

Estimation of ROS using DCFH-DA is a rapid and sensitive method [11 (link)]. After 24 h treatment, the oxidation-sensitive dye DCFH-DA (5 mg/ml) was added to L132 and L02 cells (4.0 × 10⁵cells/well) and incubated for 30 min. The cells were then collected and the intensity of the fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using Hidex plate chameleon TM V (Finland). ROS levels were calculated based on the intensity of fluorescence and expressed as percent control. For imaging, overnight grown cells on poly-L-lysine coated chambers were treated as described above. After treatment, cells were stained with DCFH-DA and images were taken using a fluorescence microscope (Olympus, Japan).

The toxic effect of oosporein on mitochondrial damage was determined by measuring the MMP using the fluorescent probe rhodamine 123. Cells were cultured in 24 well plates for fluorimetric analysis. After the toxin treatments, rhodamine 123 (10 μg/mL) was added to the cells and incubated for 1 h at 37°C. After washing twice with PBS, the cells were collected and the fluorescence was detected at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using Hidex plate chameleon^TM V (Finland).