Evaluation of Reactive Oxygen Species (ROS) Generation in Cells Using DCFDA

December 03, 2015

BlueskyReddit
George Washington University, Washington DC
Surgery
Post-doctoral Fellow

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Company:

Thermo Fisher Scientific

Product Name:

H2DCFDA (H2-DCF, DCF)/ DCF-DA

Catalog Number:

D-399

Cellular ROS levels can be measured in live cells by a technique that converts 2’,7’ –dichlorofluorescin diacetate (DCFDA) which is oxidized to a fluorescence dye 2’, 7’ –dichlorofluorescein (DCF). The fluorescence generated is directly proportional to the amount of oxidized DCFDA to DCF. Since the emission of the fluorescent dye is about 529nm, it can be measured in the FL-1 green channel by flow cytometry or fluorescent microscopy.

Experimental Design and Results Summary

Application

Flow cytometry

Starting Material

T cells (basal and activated)

Protocol Overview

T cells were either stimulated (a-CD3/aCD28) or were left unstimulated (basal). A third group had cells which would not be stained with DCFDA and provide a control. Both the stimulated and basal cells were subjected to staining. DCF-DA is primarily reconstituted with either DMSO or 100% ethanol to a stock concentration of 1mM. The working concentration is 1uM which is achieved by diluting the stock 1:1000 in PBS. Cells were incubated with the working concentration of DCFDA for 30 mins at 4 deg. CNext cells were washed twice with PBS and resuspended in FACS buffer. MFI was measured for unstained, stimulated and basal group in the FL-1/green channel on  a flow cytometer.

Tips

The working concentration may range from 1-10uM and incubation time may vary slightly (5-60 min.) DMSO at a slightly higher concentration is detrimental to cells. Thus the stock solution, which is dissolved in DMSO, has to be diluted about 1000 fold to avoid any effects of DMSO itself on cells.

Results Summary

Great staining was achieved which showed definitive increase in DCF fluorescence (FL-1) in the activated compared to basal cells, showing an increase in cytosolic ROS generation.

Additional Notes

Make small aliquots of stock solution for DCFDA

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Summary

The Good

Good protocol for measurement of ROS generation in live cells. Even small changes in ROS levels can be detected by corresponding increase in DCF fluorescence (MFI)

The Bad

This protocol only works in live cells as DCFDA is first converted to a non-fluorescent intermediate by cellular esterases which is then oxidized to florescenct DCF.

The Bottom Line

Great experimental method to detect cytosolic ROS generation if protocol is followed stringently.

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