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Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit*Optimized for Flow Cytometry*

Detection of intracellular ROS in Jurkat cells upon TBHP treatment using Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit. Cells were incubated with Amplite™ ROS Green at 37 °C for 1 hour. The cells were then treated without (Green) or with (Red) 100 µM TBHP at 37 °C for 30 minutes. The fluorescence signal was monitored at FITC channel using a flow cytometer (Acea NovoCyte 3000).
Detection of intracellular ROS in Jurkat cells upon TBHP treatment using Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit. Cells were incubated with Amplite™ ROS Green at 37 °C for 1 hour. The cells were then treated without (Green) or with (Red) 100 µM TBHP at 37 °C for 30 minutes. The fluorescence signal was monitored at FITC channel using a flow cytometer (Acea NovoCyte 3000).
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H-phraseH303, H313, H333
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OverviewpdfSDSpdfProtocol


Reactive oxygen species (ROS) are natural byproducts of the normal metabolism of oxygen and play important roles in cell signaling. However, during oxidative stress-related states, ROS levels can increase dramatically. The accumulation of ROS results in significant damage to cell structures. The role of oxidative stress in cardiovascular disease, diabetes, osteoporosis, stroke, inflammatory diseases, a number of neurodegenerative diseases and cancer has been well established. The ROS measurement will help to determine how oxidative stress modulates varied intracellular pathways. Cell Meter™ Fluorimetric ROS Assay Kit uses our unique Amplite™ ROS Green sensor to quantify ROS in live cells. Amplite™ ROS Green is cell-permeable. It generates the green fluorescence when it reacts with ROS. The Cell Meter™ Fluorimetric ROS Assay Kit provides a sensitive, one-step fluorimetric assay to detect intracellular ROS in live cells with one hour incubation. This kit is optimized for flow cytometry applications, its signal can be detected with Ex/Em = 490/520 nm (FL1 channel).

Platform


Flow cytometer

Excitation488 nm laser
Emission530/30 nm filter
Instrument specification(s)FITC channel

Components


Component A: Amplite™ ROS Green1 vial
Component B: Assay Buffer1 bottle (10 mL)
Component C: DMSO1 vial (200 µL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells at the density of 0.5 - 1 × 106 cells/mL
  2. Add 1 µL 500X Amplite™ ROS Green into 0.5 mL cell suspension
  3. Stain the cells at 37 ºC for 1 hour
  4. Treat the cells to induce ROS
  5. Analyze cells using flow cytometer with FL1 channel (Ex/Em = 490/520 nm)

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. Amplite™ ROS Green stock solution (500X):
Add 100 µL of DMSO (Component C) into the vial of Amplite™ ROS Green (Component A) and mix well to make 500X Amplite™ ROS Green stock solution. Protect from light.  Note: For storage, seal tubes tightly.

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

SAMPLE EXPERIMENTAL PROTOCOL

  1. For each sample, prepare cells in 0.5 mL Assay Buffer (Component B) or buffer of your choice at a density of 5×105 to 1×106 cells/mL. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for ROS induction

  2. Add 1 µL of 500X Amplite™ ROS Green stock solution into 0.5 mL cell suspension.

  3. Incubate at 37 ºC for 1 hour. Note: For adherent cells, gently lift the cells with 0.5 mM EDTA to keep the cells intact, and wash the cells once with serum-containing media prior to incubation with Amplite™ ROS Green. The appropriate incubation time depends on the individual cell type and test compound used. Optimize the incubation time for each experiment.

  4. Treat cells by adding 50 µL of 11X test compounds in the desired buffer (such as PBS or HBSS). For control wells (untreated cells), add the corresponding amount of buffer. 

  5. Incubate the cells at 37 ºC for a desired period of time to induce ROS, protected from light. Note: We treated Jurkat cells with 100 µM TBHP (tert-Butyl hydroperoxide) at 37 ºC for 30 minutes to induce ROS. See Figure 1 for details.

  6. Monitor the fluorescence intensity using a flow cytometer with FL1 channel (Ex/Em = 490/520 nm). Gate on the cells of interest, excluding debris.

Citations


View all 12 citations: Citation Explorer
Ginsenoside Rb1 attenuates high glucose-induced oxidative injury via the NAD-PARP-SIRT axis in rat retinal capillary endothelial cells
Authors: Fan, Chunlan and Ma, Qing and Xu, Meng and Qiao, Yuan and Zhang, Yi and Li, Pin and Bi, Yucong and Tang, Minke
Journal: International journal of molecular sciences (2019): 4936
Danazol mediates collateral sensitivity via STAT3/Myc related pathway in multidrug-resistant cancer cells
Authors: Chang, Ying-Tzu and Teng, Yu-Ning and Lin, Kun-I and Wang, Charles CN and Morris-Natschke, Susan L and Lee, Kuo-Hsiung and Hung, Chin-Chuan
Journal: Scientific reports (2019): 1--11
Notoginsenoside R1 attenuates high glucose-induced endothelial damage in rat retinal capillary endothelial cells by modulating the intracellular redox state
Authors: Fan, Chunlan and Qiao, Yuan and Tang, Minke
Journal: Drug design, development and therapy (2017): 3343
Notoginsenoside R1 attenuates high glucose-induced endothelial damage in rat retinal capillary endothelial cells by modulating the intracellular redox state
Authors: Fan, Chunlan and Qiao, Yuan and Tang, Minke
Journal: Drug Design, Development and Therapy (2017): 3343
Anti-proliferation effect of blue light-emitting diodes against antibiotic-resistant Helicobacter pylori
Authors: Ma, Jianwei and Hiratsuka, Takahiro and Etoh, Tsuyoshi and Akada, Junko and Fujishima, Hajime and Shiraishi, Norio and Yamaoka, Yoshio and Inomata, Masafumi
Journal: Journal of Gastroenterology and Hepatology (2017)
Good hydration and cell-biological performances of superparamagnetic calcium phosphate cement with concentration-dependent osteogenesis and angiogenesis induced by ferric iron
Authors: Zhang, J and Shi, HS and Liu, JQ and Yu, T and Shen, ZH and Ye, JD
Journal: Journal of Materials Chemistry B (2015): 8782--8795
Topiramate Protects Pericytes from Glucotoxicity: Role for Mitochondrial CA VA in Cerebromicrovascular Disease in Diabetes
Authors: Patrick, Ping and Price, Tulin O and Diogo, Ana L and Sheibani, Nader and Banks, William A and Shah, Gul N
Journal: Journal of endocrinology and diabetes (2015)
Superoxide dismutase as a target of clioquinol-induced neurotoxicity
Authors: Kawamura, Kazuyuki and Kuroda, Yukiko and Sogo, Masako and Fujimoto, Miki and Inui, Toshio and Mitsui, Takao
Journal: Biochemical and biophysical research communications (2014): 181--185
Down-regulated peroxisome proliferator-activated receptor γ (PPARγ) in lung epithelial cells promotes a PPARγ agonist-reversible proinflammatory phenotype in chronic obstructive pulmonary disease (COPD)
Authors: Lakshmi, Sowmya P and Reddy, Aravind T and Zhang, Yingze and Sciurba, Frank C and Mallampalli, Rama K and Duncan, Steven R and Reddy, Raju C
Journal: Journal of Biological Chemistry (2014): 6383--6393
Xanthine oxidase inhibition by febuxostat attenuates experimental atherosclerosis in mice
Authors: Nomura, Johji and Busso, Nathalie and Ives, Annette and Matsui, Chieko and Tsujimoto, Syunsuke and Shirakura, Takashi and Tamura, Mizuho and Kobayashi, Tsunefumi and So, Alex and er , undefined and Yamanaka, Yoshihiro
Journal: Scientific reports (2014): 4554

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