Hey Sravya,

your protocol sounds fine to me. Just a few suggestions.

After the staining and washing, do not wash the cells again. Since this DCF is highly diffusable you will loose more and more dye, which diffuses back into the extracellular medium. Also add any kind of stimulus after your final volume was applied. Moreover, please do not only use H2O2. This is like you would put IL-6 in your IL-6 ELISA and of course you will get a signal. You can try any kind of physioligcal stimulus relevant for your project or PMA as positive control.

For any further questions, please feel free to ask any time. And if you like also have a look at our small review:

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).

Article Functions of ROS in Macrophages and Antimicrobial Immunity

All the best and stay healthy,

Marc

Hello, Sravya!

Plese You look at following articles:

Article Photodamage attenuation effect by angelic acid in UVA irradi...

The NHDF derived from human skin cells were obtained from the Lonza Group (Basel, Switzerland) and were maintained in DMEM

DCFH-DA scavenging assay

The intracellular ROS generation was measured using the fluorescence dye DCFH-DA, which produces a detectable fluorescence when the non-fluorescent DCFH reacts with ROS in cells. The treated cells were incubated with 25 μM DCFH-DA at 37 °C for 1 h, and then the fluorescence intensity was measured using a BD fluorescence-activated cell sorting calibur (FACSCalibur, flow cytometer, BD Biosciences, San Jose, CA, USA), at excitation and emission wavelengths of 485 and 535 nm, respectively.

Article Protective properties of geniposide against UV-B-induced pho...

Cell culture

A human dermal fibroblast cell line, CCD986Sk (ATCC, Manassas, VA), was grown in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% heat-inactivated foetal bovine serum (FBS), 100 μg/mL streptomycin and 100 units/mL penicillin in a humidified atmosphere with 5% CO2 at 37 °C.

Quantitation of intracellular ROS

An ROS-sensitive probe DCFH-DA, which generates the fluorescent 2′,7′-dichlorofluorescein (DCF; λexcitation = 485 nm, λemission = 530 nm) upon enzymatic reduction and subsequent oxidation by ROS, was used (Royall and Ischiropoulos 1993). Two other ROS probes, such as DHE and DHR-123, were also used in a similar manner, which produce fluorescent 2-hydroxyethidium (2-HE; λexcitation = 480 nm, λemission = 525 nm) and rhodamine 123 (RH-123; λexcitation = 500 nm, λemission = 535 nm), respectively, upon reaction with ROS. After cells were treated with geniposide and/or 20 μM DCFH-DA, 5 μM DHE or 5 μM DHR-123 for 30 min at 37 °C, they were twice washed with 1 mL FBS-free DMEM. The cells, resuspended in 1 mL FBS-free DMEM, were irradiated. The ROS levels were quantitated by monitoring fluorescence using multi-mode microplate reader (Synergy™ Mx, BioTek Instruments, Winooki, VT).

You try to increase the dose 20 μM DCFH-DA

Hello Alexandr, Thank you for your suggestion I will increase my dose of DCFH2-DA. Is there any protocol to measure the ROS levels by using fluorescence microscope?

Thank you Marc,

I have a question? After using the dye if we are not going to wash how can we remove the excess unlabeled dye?

Hello Alexandr, I have one more question.

-In the second article, they used the dye before or after the UVB irradiation?

-For quantification of ROS levels (The cells, resuspended in 1 mL FBS-free DMEM, were irradiated. The ROS levels were quantitated by monitoring fluorescence using multi-mode microplate reader) they used the resuspended cell media directly into the 96well plate or they added cells lysate and Bradford reagent to that cells before using the micro plate reader?

Hey Sravya,

you wash your cells after the DCF staining (one wash step is enough), that is right. But you do not wash your cells after you added your final HBSS volume or any kind of stimulus (in your case H2O2).

All the best,

Marc