Cell Meter™ Fluorimetric Mitochondrial Superoxide Activity Assay KitOptimized for Microplate Reader

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12352200

Alternative formats

Cell Meter™ Fluorimetric Mitochondrial Superoxide Activity Assay Kit *Green Fluorescence*

Cell Meter™ Fluorimetric Mitochondrial Superoxide Activity Assay Kit*Optimized for Flow Cytometry*

Overview SDS Protocol

Platform

Fluorescence microplate reader

Excitation	540 nm
Emission	590 nm
Cutoff	570 nm
Recommended plate	Black wall/clear bottom
Instrument specification(s)	Bottom read mode

Components

Example protocol

AT A GLANCE

Protocol summary

Prepare cells in growth medium
Treat the cells with test compounds to induce superoxide
Add MitoROS™ 580 working solution
Stain the cells at 37°C for 30 - 60 minutes
Monitor the fluorescence increase (bottom read mode) at Ex/Em= 540/590 nm (Cutoff = 570 nm) or fluorescence microscope with TRITC filter set

Important notes
Thaw all the components at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. MitoROS™ 580 stock solution (500X):
Add 50 µL of DMSO (Component C) into the vial of MitoROS™ 580 (Component A) and mix well to make 500X MitoROS™ 580 stock solution. Protect from light. Note: 25 µL of 500X MitoROS™ 580 stock solution is enough for 1 plate. For storage, seal tubes tightly.

PREPARATION OF WORKING SOLUTION

Add 25 μL of 500X MitoROS™ 580 stock solution into 10 mL of Assay Buffer (Component B) and mix well to make MitoROS™ 580 working solution. Note: This MitoROS™ 580 working solution is stable for at least 2 hours at room temperature.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

SAMPLE EXPERIMENTAL PROTOCOL

Treat cells with 10 µL of 10X test compounds (96-well plate) or 5 µL of 5X test compounds (384-well plate) in your desired buffer (such as PBS or HHBS). For control wells (untreated cells), add the corresponding amount of compound buffer.
To induce superoxide, incubate the cell plate at 37°C for a desired period of time, protect from light. Note: We treated HeLa cells with 50 µM Antimycin A (AMA) at 37°C for 30 minutes to induce superoxide. See Figure 1 for details.
Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of MitoROS™ 580 working solution into the cell plate.
Incubate the cells at 37°C for 30 to 60 minutes.
Monitor the fluorescence increase with a fluorescence microplate reader (bottom read mode) at Ex/Em = 540/590 nm (Cutoff = 570 m) or observe cells using a fluorescence microscope with TRITC filter.

Images

Figure 1. Fluorescence images of superoxide measurement in HeLa cells using Cell Meter™ Fluorimetric Intracellular Superoxide Detection Kit (Cat#22971). HeLa cells at 100,000 cells/well/100 µL were seeded overnight in a 96-well black wall/clear bottom plate. AMA Treatment: Cells were treated with 50 µM Antimycin A (AMA) at 37 °C for 30 minutes, then incubated with MitoROS™ 580 for 1 hour. Untreated Control: HeLa cells were incubated with MitoROS™ 580 at 37 °C for 1 hour without AMA treatment. The fluorescence signal was measured using fluorescence microscope with a TRITC filter.

Figure 2. Detection of intracellular superoxide in HeLa cells using Cell Meter™ Fluorimetric Intracellular Superoxide Detection Kit (Cat#22971). HeLa cells at 100,000 cells/well/100 µL were seeded overnight in a 96-well black wall/clear bottom plate. Cells were incubated with 50 µM Pyocyanin (Pyo); 50 µM Antimycin A (AMA) or without treatment (Control) at 37 ºC for 30 minutes. Cells were then incubated with MitoROS™ 580 at 37 ºC for 1 hour. The fluorescence signal were monitored at Ex/Em = 540/590 nm (Cutoff = 570 nm) with bottom read mode using a CLARIOstar microplate reader (BMG Labtech).

Citations

View all 4 citations: Citation Explorer

GRP75-faciliated Mitochondria-associated ER Membrane (MAM) Integrity controls Cisplatin-resistance in Ovarian Cancer Patients
Authors: Li, Jing and Qi, Fangzheng and Su, Huishan and Zhang, Chuanshan and Zhang, Qing and Chen, Ying and Chen, Ping and Su, Linjia and Chen, Yanan and Yang, Yuqi and others,
Journal: International journal of biological sciences (2022): 2914

High-glucose-induced apoptosis, ROS production and pro-inflammatory response in cardiomyocytes is attenuated by metformin treatment via PP2A activation
Authors: Cheng, Gang and Li, Lihuan
Journal: Journal of Biosciences (2020): 1--11

Effect of antioxidants on the H2O2-induced premature senescence of human fibroblasts
Authors: Pie{\'n}kowska, Natalia and Bartosz, Grzegorz and Pichla, Monika and Grzesik-Pietrasiewicz, Michalina and Gruchala, Martyna and Sadowska-Bartosz, Izabela
Journal: Aging (Albany NY) (2020): 1910

Nitroxide Radical-Containing Redox Nanoparticles Protect Neuroblastoma SH-SY5Y Cells against 6-Hydroxydopamine Toxicity
Authors: Pichla, Monika and Pulaski, {\L}ukasz and Kania, Katarzyna Dominika and Stefaniuk, Ireneusz and Cieniek, Bogumi{\l} and Pie{\'n}kowska, Natalia and Bartosz, Grzegorz and Sadowska-Bartosz, Izabela
Journal: Oxidative Medicine and Cellular Longevity (2020)

References

View all 60 references: Citation Explorer

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Journal: J Toxicol Sci (2014): 71

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