Cellrox deep red oxidative stress reagent

Cells after TA exposure were stained with CellROX Deep Red oxidative stress reagent (5 μM; Life Technologies, Carlsbad, CA, USA) and subjected for run on an Accuri C6 Flow Cytometer (BD Biosciences, San Jose, CA, USA). The obtained data was interpreted through using BD accuri C6 software (BD Biosciences). More than 10000 mononuclear cells were chosen in each run within the gated channel drawn in forward and side scatter plot and analyzed for mean fluorescence intensity. The extent of ROS production was ascertained by staining of cells with CellROX Deep Red oxidative stress reagent (5 μM; Life Technologies, Carlsbad, CA, USA)^{69 (link)}. An Accuri C6 Flow Cytometer (BD Biosciences, San Jose, CA) was employed to evaluate the ROS fluorescence levels.
Also, cells were seeded 1 × 10⁴ in 4-chambered slides (Sarstedt Inc., Newton, NC, USA) and treated using 10 and 20 µM TA. Post 24 h exposure, treated cells were stained with CellROX Deep Red reagent and fixed. Further, cells were permeabilized and nuclei counterstained using a DAPI stain, mounted and imaged using confocal laser scanning microscopy (Carl Zeiss LSM 710, Oberkochen, Germany) at 40X magnification^{63 (link)}.

The reagent used for this assay was a fluorogenic probe to measure cellular oxidative stress upon oxidation by reactive oxygen species (ROS). To perform the test, cells were incubated with the compounds to be tested, at IC₉₀ for 24 h. The cells were centrifuged and resuspended in a volume of 50 μl, following which CellROX™ Deep Red oxidative stress reagent (Thermo-Fisher Scientific) was added to 96-well plates at 5 mM. The plates were then incubated for 30 min at 37 °C. After incubation, the results were analyzed by cell imaging using the Evos cell imaging system. Cells treated with different compounds were compared with untreated control cells and the positive control. Fluorescence intensity was also measured using the Enspire multimode plate reader at excitation/emission wavelengths of 640/665 nm.

Intracellular ROS level was measured by use of fluorescent dye dichlorofluorescin diacetate (H₂DCFDA), a nonpolar compound that is converted into a polar derivative (dichlorofluorescein) by cellular esterase after incorporation into cells [16 (link)]. On the day of the experiment, the medium was replaced with Hank’s solution containing 10 µM H₂DCFDA dye and cells were incubated. Following 30 min later, intracellular fluorescence was detected with excitation at 485 nm and emission at 530 nm by a Spectra Max Gemini EM (Mol. Devices, Sunnyvale, CA, USA).
ROS level were measure according to the company’s protocol (CellROX Deep Red Oxidative Stress Reagent, Catalog No. C10422, Thermo Scientific, Carlsbad, CA, USA). In brief, LECs (5 × 10³) transfected with pEGFP-Vector or pEGFP-Sumo1 with pCl-neo-HA-Sp1 or pCl-neo-HA-Sp1K16R cultured in 96-well plate, 48 h later cells were exposed with different concentration of H₂O₂. After 8h, CellROX deep red reagent was added with final concentration of 5μM and cells were incubated at 37°C for 30 min. Media containing CellROX deep red reagent were removed and fixed with 3.7% formaldehyde. After 15 min, fluorescence signal were measured at Ex640 nm/ Em665 nm [12 (link)].