Modulus

The ROS detection assay was performed with 2′,7′-dichlorofluorescin diacetate (DCFDA) staining (Sigma). Briefly, cells were seeded in triplicate at a density of 0.2 × 10⁵ cells/well in opaque flat bottom 96-well tissue culture plates in 100 μL media without phenol red and allowed to grow for 18 h. Following transfection and/or treatments, cells were washed with PBS and maintained in 100 μL of phenol red-free medium and further incubated for 24 h. A 50 mM stock solution of DCFDA was added to each well containing 100 μL pre-existing media to achieve a final concentration of 25 μM and incubated for 45 min at 37°C. Fluorescence signal intensities indicating ROS levels were recorded by taking readings using a 96-well fluorescent multiplate reader (MODULUS, Promega) using excitation and emission spectra of 485 nm/535 nm. To normalise the fluorescence signal, cells in the same wells were stained with Coomassie brilliant blue stain (Sigma) for 1 h and washed with distilled water and 10% sodium dodecyl sulphate (SDS) solution was added to release the absorbed dye for 10 min while shaking. The absorbance values at 595 nm were then recorded using a multiplate absorbance reader (MODULUS, Promega) and the data was used after normalising the fluorescence values.

For measurement of ROS, cells (2 × 10⁴/mL) were plated in triplicate in opaque 96-well plates in phenol red-free medium (100 mL). After 18 h, an equal volume (100 mL) of a 2’,7’-dichlorofluorescein diacetate (DCFDA, 50 mM) solution was added to each well and left to react for 45 min at 37°C. ROS levels were measured by fluorescence using excitation (485 nm) and emission (535 nm) spectra in a multiplate reader (MODULUS, Promega). For normalization, cells were stained with Coomassie blue stain (Sigma) for 1 h then washed with distilled H₂O. To release the dye, sodium dodecyl sulphate (10% solution) was added with 10 min shaking. Absorbance was read at 595 nm in the above multiplate reader.
For measurement of glutathione, cells (1 × 10⁴/mL) were plated in white clear bottom 96-well plates and left for 18 h. Media were removed from the wells. The glutathione (GSH)/GSSG-Glo™ Assay (Promega) was used according to the manufacturer’s protocol. Total glutathione lysis reagent (50 mL) was added to each well and lysates were transferred to a 96-well opaque plate. Plates were shaken at room temperature for 5 min. Luciferin Generation Reagent (50 mL) was added to each well and plates shaken and left at room temperature for 30 min. Luciferin Detection Reagent (100 mL) was added and plates were shaken for 15 min. Luminescence was then measured using a luminometer (MODULUS, Promega).

ROS detection assay was performed by using 2′,7′-Dichlorofluorescin diacetate (DCFDA) staining (Sigma). Briefly, cells were seeded in triplicate at a density of 0.2 × 10⁵ cells/well of opaque flat bottom 96-well tissue culture plates in 100 μL media without phenol red and allowed to grow for 18 h. Following transfection, cells were washed with PBS and maintained in 100 μL of phenol red-free medium and further incubated for 24 h. A 50 mM stock solution of DCFDA was added to each well containing 100 μL preexisting media to achieve a final concentration of 25 μM and incubated for 45 min at 37°C. Fluorescence signal intensities indicating ROS levels were recorded by taking readings using 96-well fluorescent multiplate reader (MODULUS, Promega) using excitation and emission spectra of 485 nm/535 nm. To normalise the fluorescence signal, cells in the same wells were stained with Coomassie brilliant blue stain (Sigma) for 1 h and washed with distilled water and 10% sodium dodecyl sulphate (SDS) solution was added to release the absorbed dye for 10 min while shaking. The absorbance values at 595 nm were then recorded using a multiplate absorbance reader (MODULUS, Promega) data used after normalising the fluorescence values.