At the day 3 post-PGPS inoculation and TUDCA treatment, the mice were sacrificed, and their right bullae were dissected and placed in 10% FBS RPMI 1640 medium in a plastic culture dish. After all non-middle ear-associated tissues were removed, the mucosa in the middle ear cavity was gently removed with fine forceps and transferred to the center of a 35-mm glass-bottom culture dish and washed three times with 1 × PBS. The samples were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature. Samples were washed twice in PBS to remove residual paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS for 15 min, and then washed three times in 1 × PBS. The specimens were incubated with a CellROX® Green Reagent kit (Invitrogen, 10444) at 37°C for 30 min, washed three times in 1 × PBS, and then counterstained with DAPI for 5 min at room temperature. The stained tissues were observed under an immunofluorescence microscope (Leica DFC500, Germany).
Cellrox green reagent kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CellROX® Green Reagent kit is a fluorogenic probe that detects reactive oxygen species (ROS) in live cells. It functions by becoming fluorescent upon oxidation, allowing for the visualization and quantification of ROS levels within the cellular environment.
Automatically generated - may contain errors
4 protocols using cellrox green reagent kit
1
Assessing Oxidative Stress in Murine Middle Ear Tissue
2
Nitric Oxide and ROS Measurement in HD11 Cells
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Nitrite (NaNO 2 ) concentration, as an index of nitric oxide (NO) production, was determined by spectrophotometry in cell culture supernatants using a standard Griess assay according to the manufacturer's instructions (Promega, UK). The absorbance was read at 550 nm in a Multiskan Ascent plate reader (Thermo Fisher Scientific, USA). The nitrite concentration was calculated using a sodium nitrite standard curve. ROS (Reactive Oxygen Species) production was evaluated by flow cytometry using the CellROX ® Green Reagent kit (Invitrogen, USA) according to the manufacturer's instructions.
Briefly, HD11 cells were seeded at 2x10 5 cells/well in 24-well plates and pretreated with IFNα (50 ng/ml) for 16h (unless otherwise indicated). Next, cells were gently washed with
Briefly, HD11 cells were seeded at 2x10 5 cells/well in 24-well plates and pretreated with IFNα (50 ng/ml) for 16h (unless otherwise indicated). Next, cells were gently washed with
3
Intracellular ROS Measurement by CellROX
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Adherent cells were seeded overnight and treated with 4 Gy IR in the culture medium. After 24 h, CellROX Green Reagent Kit (Thermo Scientific) was used to detect intracellular ROS levels. Briefly, CellROX Green reagent was added to the cells with the final concentration of 5 × 10−6m and 800 × 10−9m for assays of microplate reader and flow cytometry, respectively, and incubated at 37 °C for 30 min in the dark. The stained cells were washed in buffer, and the fluorescence intensity was measured using the Infinite M200 PRO microplate reader (Tecan, Männedorf, Switzerland) at excitation/emission = 485/520 nm or flow cytometer (LSR Fortessa X‐20, BD Bioscience, CA, USA) at the 450/50 bandpass filter.
4
Measuring Cellular Oxidative Stress Levels
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