Measuring Intracellular ROS in H358 Cells

The generation of ROS in H358 cells was measured using a ROS sensitive fluorescent probe, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, ThermoFisher). DCFH-DA can be oxidized to 2′,7′-dichlorofluorescein (DCF) by ROS and exhibits green fluorescence intensity.⁸⁰ H358 cells were seeded on 25 × 25 mm microscope cover glass slips in BD Falon 60 × 60 mm tissue culture dishes for 72 h. Untreated cells were maintained as the negative controls whereas 10, 20 and 30% H₂O₂ solution in PBS was administered to cells for 15 minutes as positive controls.^{54 (link)} H358 cells were also dosed with IC₅₀ values of various complex as follows: 4⁴⁺ (15 μM), 3³⁺ (13 μM) and 2²⁺ (1.7 μM) for 3 time periods of 2, 8, and 22 h. The cells were passaged and washed 3× in ice cold PBS then suspended in 10 mM DCFH-DA in PBS and incubated in the dark for 30 min. The levels of intracellular ROS were examined by confocal microscopy using long pass light filters and a 1.3 airy unit pinhole at 488/529 nm with a Zeiss axio-plane inverted fluorescence microscope.