The 2’,7’ –dichlorofluorescin diacetate (H2DCFDA) is a fluorescent probe that we commonly use in our labs to measure the level of ROS generation. This is acylated derivative the easily enter cells and deacylated by esterase enzymes in the cells. Upon reaction with ROS, a fluorescent dye is generated that you can quantify by fluorescence spectroscopy. The good thing about the method is that you only need 30-45 minutes to do the staining and hence you add the H2DCFDA during the last 30-45 min of the assay. Unless your incubation of the cells with the drug is for less than an hour, you will then add the fluorescent probe after the drug/treatment.

Hey Rashmi,

Solomon already explained it very well. Only a few suggestions. In the final medium you should not have any kind of serum or phenol red, because it highly influences and falsifies your fluorescence signal. You can use any kind of clear full medium.

The difficulty as always with ROS measurements is the questions: does your drug treatment induces a quick or a slow ROS response or a ROS response at all? Therefore it is hard to tell, when you should stain your cells. I would try both options: Stain before and after drug treatment.

H2DCF-DA/DCF-DA is a derivate of fluorescein, which is cell-permeable and one off the most commonly used ROS probes. Once in the cytosol the two acetate groups get cleaved by cellular esterases, generating 2',7'-Dichlordihydrofluorescein. This molecule can now be oxidized by ROS to generate the fluorescent 2’, 7’ –dichlorofluorescein. This ROS probe is often termed as intracellular ROS probe, but after cleavage it remains highly diffusible, reaching not only organelles and phagosomes but also diffuses into the extracellular space. Therefore only total cellular ROS can be detected with this probe (Ushijima et al., 1997, Hempel et al., 1999).

5-(und -6)-Carboxy-2',7'-Dichlorofluorescein-Diacetat (6-Carboxy-DCF) is a derivate of H2DCF-DA that contains two additional carboxyl groups, enhancing its hydrophilicity and therefore highly increases its retention in the cytosol. This ROS probe can be used to specifically detect cytosolic ROS levels in contrast to H2DCF-DA, which detects total cellular ROS levels (Wolf et al., 2020, Herb et al., 2019, Mak et al., 2017, Hempel et al., 1999).

It depends, if you want to detect total cellular ROS or cytosolic ROS.

For a detailed protocol for DCF staining, please have look at:

Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO, 2019, Science Signaling 12(568):eaar5926

DOI: 10.1126/scisignal.aar5926

All the best and stay healthy.

Marc

In context of this topic, I would like to bring to your attention a recent review of our group, "Functions of ROS in macrophages and antimicrobial immunity".

In this review, we give an introduction to ROS and their sources in macrophages, summarize the versatile roles of ROS in direct and indirect antimicrobial immune defense and provide an overview of commonly used ROS probes, ROS source inhibitors and ROS scavengers (also the difference between ROS scavengers and antioxidants, which are not synonymous, is explained).

If you like, please have a look at:

Functions of ROS in Macrophages and Antimicrobial Immunity

February 2021, Antioxidants 10(2):313

DOI: https://doi.org/10.3390/antiox10020313