Works Alright with Strong Oxidative Stressors, Lower Sensitivity

Princeton
Molecular Biology
Graduate Student

Overall

Quality of Results

Ease-of-Optimization

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Company:

Abcam

Product Name:

DCFDA / H2DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit

Catalog Number:

ab113851

This kit is not much of a kit. It contains the DCFDA reagent, already reconstituted, an oxidative stress inducer, and a diluting buffer. Given that you will put the reagent into cell culture, you don't need the dilution buffer. Also, the carboxy-methyl-DCFDA reagent can be purchased for a better price at other suppliers and is retained in the cell for longer. Finally, you can use peroxide or multiple other positive controls. Overall, this kit will work for very strong inducers, but testing in standard breast cancer cell lines with effective doses of chemotherapy showed no signal above the variable background. I recommend purchasing cm-DCFDA or dihydroethidium.

Experimental Design and Results Summary

Application

Flow cytometry

Starting Material

Human breast cancer cells

Protocol Overview

Product is added to cell culture at 20 µM for 4–24 hr. Appropriate positive and negative controls such as n-acetylcysteine and peroxide are used. Cells are washed, trypsinized, and measured by flow in the FITC channel.

Tips

Order dry carboxy-methyl DCFDA from other suppliers for cheaper and more robust results.

Results Summary

Showed positive signal for strong oxidative conditions, but weak signal overall.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

Very easy to use

The Bad

Low sensitivity

The Bottom Line

Try other reporters such as cm-DCFDA first. They are cheaper and more robust.

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