hDASMCs were plated at an experimentally determined, optimal density for fluorescence imaging in a μ‐Slide 8 Well Glass Bottom plate (part number 80827; Ibidi, Fitchburg, WI). The next day, cells were loaded with 10 μmol/L of MitoROS 580 dye using the Mitochondrial Superoxide Detection Kit (ab219943; Abcam). MitoROS was incubated with the cells for 30 minutes at 37 °C in a hypoxic incubator, in the presence of either DMSO, 50 μmol/L S1QEL, 100 μmol/L S3QEL, or 10 μmol/L rotenone. After loading, cell chambers were placed in an OkoLab stage‐top microscope incubator, and imaging was performed using a Leica TCS SP8 X confocal microscope (excitation 540 nm, emission 570–720 nm, 2.5 frames/second, Leica Microsystems). Cells were allowed to equilibrate in hypoxia for 10 minutes, before imaging for 10 minutes in hypoxia followed by 10 minutes of normoxia. Images were obtained using LAS‐X software (Leica) and representative images made using Fiji (ImageJ). For each treatment group, ROI were drawn around cells to track changes in MitoROS fluorescence over time. To compare changes in MitoROS fluorescence induced by increased O2 content, ROI intensity over the last 4 images of normoxic incubation were compared with the initial 4 images taken during hypoxic incubation.
Mitochondrial superoxide detection kit
Manufactured by Abcam
Sourced in United Kingdom
The Mitochondrial Superoxide Detection Kit is a fluorometric assay designed to detect and quantify superoxide levels in the mitochondria of cells. The kit utilizes a mitochondria-targeted fluorogenic probe that specifically reacts with superoxide, producing a fluorescent signal that can be measured.
Automatically generated - may contain errors
Lab products found in correlation
Tcs sp8 x confocal microscope, by Leica (1 mentions)
Las x software, by Leica (1 mentions)
μ slide 8 well glass bottom plate, by Ibidi (1 mentions)
Ab113851, by Abcam (1 mentions)
A22188, by Thermo Fisher Scientific (1 mentions)
Varioskan flash, by Thermo Fisher Scientific (1 mentions)
Infinite m200 microplate reader, by Tecan (1 mentions)
5 protocols using mitochondrial superoxide detection kit
1
Hypoxia-Induced Mitochondrial Oxidative Stress
2
Mitochondrial Superoxide Assay in Murine Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mitochondrial superoxide was measured in bone marrow cells and splenocytes of mice (isolated immediately after the sacrifice) using Mitochondrial Superoxide Detection Kit (Abcam, Cambridge, UK), a sensitive fluorometric one-step assay based on MitoROS 580 dye to detect intracellular superoxide radical in live cells. The dye is cell-permeable and selectively reacts with mitochondrial superoxide present in live cells to generate a red fluorescence signal that was read at Ex/Em = 540/590 nm in a microplate reader after incubation at 37 °C for 60 min. The results were calculated as fluorescence intensity difference between control and treated cells. Data are expressed as M.F.I. (a.u.).
3
Measurement of Cellular ROS and H2O2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Reactive oxygen species (ROS) were measured using the cell permeant reagent 2′,7′‐dichlorofluorescin diacetate (DCFDA) according to the manufacturer's protocol (Abcam, ab113851). DCFDA is deacetylated by cellular esterases and oxidized by ROS into a highly fluorescent compound which was measured using a fluorescence microplate reader (excitation/emission wavelength of 488/535 nm). Amplex® Red reagent (10‐acetyl‐3,7‐dihydroxyphenoxazine) was used to detect hydrogen peroxide (H2O2). In the presence of peroxidase, the Amplex® Red reagent reacts with H2O2 to produce the red‐fluorescent oxidation product, resorufin, which was measured using fluorescence microplate reader (excitation/emission wavelength of 430/590 nm), according to the manufacturer's protocol (A22188, Invitrogen). To detect production of mitochondrial superoxide radical in live cells, the Mitochondrial Superoxide Detection Kit (ab219943, Abcam) was used, according to the manufacturer's protocols. Superoxide was measured using fluorescence microplate reader (excitation/emission wavelength of 540/590 nm).
4
Detecting Mitochondrial ROS in Apoptosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Changes in mitochondrial ROS that occurred during apoptosis were detected with a fluorescence-based assay. Mitochondrial ROS was detected with a Mitochondrial Superoxide Detection Kit (ab219943, Abcam, Cambridge, United Kingdom). MCF-7 cells were cultured as previously described. After being starved with 0.25% stripped FBS-containing medium for 2 days, cells were re-fed using 5% stripped FBS-containing medium and treated with E2 and heteronemin. Antimycin A at 50 μM was used as a positive control. After 24 h, cells were processed with the Mitochondrial Superoxide Detection Kit according to the manufacturer’s instructions. Excitation at 540 nm and emission at 590 nm was read with a spectral scanning multimode reader (Thermo Fisher Scientific Varioskan Flash, Waltham, MA, United States).
5
Mitochondrial Superoxide Detection Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The assay was performed using a mitochondrial superoxide detection kit (Abcam, Cambridge, UK) according to the manufacturer’s protocol. The cells were cultured in 96-well microwell dishes for 24 h and further incubated in 100 μL medium containing M4N (40 μM) and/or TMZ (30 μM) for an additional 2, 4, 24, and 48 h. Then 100 μL MitoROS 580 reagent, a fluorescence indicator for superoxide (a 500X stock solution diluted in the assay buffer), was added to each well. The cells were further incubated at 37°C for 60 min, and the fluorescence was measured at an excitation wavelength of 540 nm and emission wavelength of 590 nm (with a cutoff at 570 nm) with the Infinite M200 Microplate Reader (Tecan, Grödig, Austria).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!