MarkerGene™ Live Cell Fluorescent Reactive Oxygen Species Detection Kit

Product ID: M1049



Unit SizePriceQuantity 
1kit
$247.39
  • Buy 5 for $197.91 each and save 21%

Availability: In stock


Description

This kit provides all necessary reagents, buffers and a detailed protocol for the detection of Reactive Oxygen Species (ROS, peroxidase activity) in live cells.

In healthy aerobic cells, ROS generation occurs at a controlled rate but under high stress conditions, its production is greatly increased, resulting in changes of many cell components including proteins and lipids.

 Fluorogenic peroxidase substrates are converted to highly fluorescent products in the presence of the enzyme and hydrogen peroxide but can be relatively unstable for use in enzyme-linked immunosorbent assays (ELISAs). By forming the diacetate derivates, intracellular applications are more efficient, whereupon the acetates are cleaved by endogenous esterases, releasing the intact substrate.  In the presence of nonspecific ROS, commonly produced during oxidative stress, the fluorescein substrate becomes oxidized, emitting green fluorescence. 

 The MarkerGeneTM Live Cell Fluorescent Reactive Oxygen Species Detection Kit utilizes a cell permeable substrate that is a reliable fluorogenic marker for ROS detection. Upon enzyme activity, a highly fluorescent dye is produced, with EX: 495nm and EM: 530 nm.  In addition, this kit also contains t-butyl hydroperoxide, a common ROS inducer, as a positive control.  

The assay kit contains enough reagents to prepare up to 250 mL of staining solution, or approximately 100 assays (12-well tissue culture plate format), or 100 x 96-well tissue culture microtiterplate staining assays.  Methods for testing various drug or inducer compounds are also described.  This assay can be adapted for high-throughput, high-content assay screening analyses.  


Technical Data
SKU M1049
Unit Size 1kit
Detection Method Fluorescence

References and Citations

Citations:

  • Fallahian F, Aghaei M, Abdolmohammadi M, Hamzeloo-Moghadam M (2016) "Molecular mechanism of apoptosis induction by Gaillardin, a sesquiterpene lactone, in breast cancer cell lines" Cell Biology and Toxicology 31(6):295–305.
  • Shahsavari Z, Karami-Tehrani F, Salami S, Ghasemzadeh M. (2015) "RIP1K and RIP3K provoked by shikonin induce cell cycle arrest in the triple negative breast cancer cell line, MDA-MB-468: necroptosis as a desperate programmed suicide pathway." Tumour Biol. epub ahead of print
  • Hamzeloo-Moghadam M, Aghaei M, Fallahian F, Jafari SM, Dolati M, Abdolmohammadi MH, Hajiahmadi S, Esmaeili S. (2015) "Britannin, a sesquiterpene lactone, inhibits proliferation and induces apoptosis through the mitochondrial signaling pathway in human breast cancer cells." Tumour Biol. 36(2):1191-8.
  • Allameh A, Ahmadi-Ashtiani H, Emami Aleagha MS, Rastegar H. (2014) "The metabolic function of hepatocytes differentiated from human mesenchymal stem cells is inversely related to cellular glutathione levels." Cell Biochem Funct. 32(2):194-200.
  • Fujiwara S, Kawano Y, Yuki H, Okuno Y, Nosaka K, Mitsuya H, Hata H. (2013) "PDK1 inhibition is a novel therapeutic target in multiple myeloma." Br J Cancer. 108(1):170-8.
  • Aghaei M, Karami-Tehrani F, Panjehpour M, Salami S, Fallahian F (2012) "Adenosine induces cell-cycle arrest and apoptosis in androgen-dependent and -independent prostate cancer cell lines, LNcap-FGC-10, DU-145, and PC3." Prostate, 72: 361–375.
  • Lee TK, O'Brien KF, Wang W, Johnke RM, Sheng C, Benhabib SM, Wang T, Allison RR. (2010) "Radioprotective effect of American ginseng on human lymphocytes at 90 minutes postirradiation: a study of 40 cases." J Altern Complement Med. 16(5):561-7.
  • Lee T-K, O’Brien KF, Wang W, Sheng C, Wang T, Johnke RM, Allison RR. (2009) "American Ginseng Modifies 137Cs-Induced DNA Damage and Oxidative Stress in Human Lymphocytes." The open nuclear medicine journal. 1(1):1-8.
  • Mitsuya H, Hata H. (2008) "Induction of cell cycle arrest and apoptosis in myeloma cells by cepharanthine, a biscoclaurine alkaloid." Int J Oncol. 33(4):807-14.
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References:

  • Lyons TA, Amouretti XF, Held PG, Naleway JJ. (2010) "Development of a Live-Cell Based Reactive Oxygen Species (ROS) Assay for use in High-Content Screening of Drug Candidates Using the BioTek Synergy Mx Microplate Reader." presented at the Society for Biomolecular Sciences 16th Annual Conference in Phoenix, AZ April 11-15, 2010. link
  • Kitabchi AE, Stentz FB, Umpierrez GE. (2004) "Diabetic ketoacidosis induces in vivo activation of human T-lymphocytes." Biochem Biophys Res Commun 315: 404-7.
  • Kumar SS, Shankar B, Sainis KB. (2004)"Effect of chlorophyllin against oxidative stress in splenic lymphocytes in vitro and in vivo." Biochim Biophys Acta 1672: 100-11. 10
  • Delogu G, Antonucci A, Moretti S, Marandola M, Tellan G, Signore M, Famularo G. (2004) "Oxidative stress and mitochondrial glutathione in human lymphocytes exposed to clinically relevant anesthetic drug concentrations." J Clin Anesth 16: 189-94.
  • Kalivendi S.V., Kotamraju S., Cunningham S., Shang T., Hillard C.J., Kalyanaraman B. (2003) “1-Methyl-4-phenylpyridinium (MPP+)-induced apoptosis and mitochondrial oxidant generation: role of transferrin-receptor-dependent iron and hydrogen peroxide.” Biochem J 371:151-64.
  • Chen CY, Wang YF, Lin YH, Yen SF. (2003) "Nickel-induced oxidative stress and effect of antioxidants in human lymphocytes." Arch Toxicol 77: 123-30.
  • Diaz G, Liu S, Isola R, Diana A, Falchi AM. (2003) "Mitochondrial localization of reactive oxygen species by dihydrofluorescein probes." Histochem. Cell Biol. 120: 319-25 .
  • Tampo Y, Kotamraju S, Chitambar CR, Kalivendi SV, Keszler A, Joseph J, Kalyanaraman B. (2003) "Oxidative stress-induced iron signaling is responsible for peroxide-dependent oxidation of dichlorodihydrofluorescein in endothelial cells: role of transferrin receptor-dependent iron uptake in apoptosis." Circ. Res. 92: 56-63.
  • Blattner JR, He L, Lemasters JJ. (2001) "Screening assays for the mitochondrial permeability transition using a fluorescence multiwell plate reader." Anal. Biochem. 295: 220-226.
  • Kehrer JP, Paraidathathu T (1992) "The use of fluorescent probes to assess oxidative processes in isolated-perfused rat heart tissue." Free Radic. Res. Commun. 16: 217-222.
  • Salata RA, Sullivan JA, Mandell GL. (1983) "Visualization of hydrogen peroxide in living polymorphonuclear neutrophils utilizing leucodiacetyl 2,7-dichlorofluorescin: photomicrographic and microphotometric studies." Trans. Assoc. Am. Physicians 96: 375-383.
  • Keston AS, Brandt R (1965)"The fluorometric analysis of ultramicro quantities of hydrogen peroxide." Anal. Biochem. 11: 1-5.
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Technical Support

Question about this product? Ask a Scientist!

We pride ourselves on the high quality of our products and want you to get the best possible results from your assays. If you have any questions about this product or need help optimizing your protocol check out the product FAQs below or ask your own question and one of our expert scientists will get back to you asap:


Question: What is the recommended volume and cell density for a 6-well plate?
The recommended volume for 6 well plates would be 3-5ml and cell density should be around 500000 cells/well.
Question: Does the reagent need to be handled in a dark room?
No, however like all fluorescent reagents it should be protected from excessive light exposure.
Question: For the kinetic reading, do you recommend to go to a maximum of 4hours or can we go longer than that?
Kinetic readings can be recorded at 30 minute intervals to determine an optimum end point for the assay.
Question: Can I fix the cells or I can only look at live samples?
Unfortunately, we have not tried to use this kit with fixed cells. But I think it should work.
Please note, it is not necessary to fix the cells prior to loading this substrate, as it will passively diffuse into live cells. Nevertheless, I don't think that the fixation step should significantly affect the activity of ROS enzymes. But the substrate and the product may become more permeable and show some diffusion (bleeding) from the fixed cells, more than with live cells. If this is the case, you might have to monitor staining and either lower the concentration of the substrate or adjust the staining time (shorter time) to modulate the possible diffusion effects.
If you are counterstaining with an antibody, it might be better to stain first with antibody, then stain with the ROS kit.
Question: I see major fluctuations when I measure ROS activity in a period from 30-45 minute intervals. For instance, if I read my 24-well plate at 30 minutes incubation it can be 30-40% different than if I incubate for 35 minutes.
The ROS detection method is kinetic, and is due to enzymatic turnover of the substrate, so there should be fluctuations with time. We weren't able to find a suitable broad-spectrum inhibitor to stop the reaction at a certain time period. I would suggest running all your assays for a set time period, if you wish to compare results. It appears from your description that the reaction is not finished at 35 min. and you might want to let the assay run longer, to give the highest fluorescence output (turnover). But again, use the same time for all assays.
Question: Should I be incubating my cells with the ROS probe at 37C?
Yes, we usually return the cells to the incubator (i.e. 37°C) prior to washing and viewing. I don't think it will matter too much, since the cells will not be recovered, but either method (room temp or 37°C) is fine, as long as you use the same procedure for all assays.
Question: Should I measure fluorescence before or after a PBS wash
Our protocol suggests washing the cells after staining, mostly because we usually examine the cells by microscopy to see the staining patterns. But I think if you are just measuring the total fluorescence (turnover) using a fluorescence microtiterplate reader, then you will get more signal if you don't wash, but read directly, since you will be measuring the total turnover, with time. Some fluorescent dye tends to "leak" from the cells with time, and that can get washed away. But in microscopy, it only adds to the "background".
Either method is fine, but it depends on your application. But whichever method you choose, you have to keep that same protocol for all assays, so that they can then be compared.