DCFDA / H2DCFDA – Cellular ROS Assay Kit

DCFDA – Cellular ROS Assay Kit / Reactive Oxygen Species Assay Kit (ab113851) uses the cell permeant reagent 2’,7’ –dichlorofluorescin diacetate (DCFDA, also known as H2DCFDA, DCFH-DA, and DCFH) to quantitatively assess reactive oxygen species in live cell samples.

DCFDA / H2DCFDA / DCFH-DA / DCFH is a fluorogenic dye that measures hydroxyl, peroxyl and other reactive oxygen species (ROS) activity within the cell. NB: DCFDA and DCFA are also available as free molecules as ab145286 (Carboxy-DCFDA N-succinimidyl ester) and ab145439 (5(6)-Carboxy-2′,7′-dichlorofluorescein).

The DCFDA assay protocol is based on the diffusion of DCFDA / H2DCFDA / DCFH-DA / DCFH into the cell. It is then deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by ROS into 2’, 7’ –dichlorofluorescein (DCF). DCF is highly fluorescent and is detected by fluorescence spectroscopy with excitation / emission at 485 nm / 535 nm.

Chinese protocol available. See Protocols section below.

DCFDA assay protocol / ROS assay protocol summary (microplate):
– collect suspension cells in tube / seed and allow attachment of adherent cells in 96-well plate
– wash in buffer
– stain with DCFDA for 30 min (suspension) / 45 min (adherent), wash with buffer
– if suspension cells, transfer to microplate
– analyze with microplate reader

DCFDA assay protocol / ROS assay protocol summary (flow cytometry):
– collect cells in tubes
– stain with DCFDA for 30 min (without washing)
– analyze with flow cytometer

DCFDA assay protocol / ROS assay protocol summary (fluorescent microscopy):
– wash adherent cells with buffer
– stain with DCFDA for 45 min
– wash in buffer
– analyze with fluorescent microscope
– maintain low light conditions to reduce photo-bleaching

Previously called DCFDA / H2DCFDA – Cellular Reactive Oxygen Species Detection Assay Kit.

This kit contains sufficient materials for approximately 300 measurements in microplate format and 70 measurements (35 mL) by flow cytometry. The number of measurements in microscopy is dependent on experimental setup.

This kit is not compatible with fixed samples. Stained cells must be measured live.

Related products

Review the oxidative stress marker and assay guide, or the full metabolism assay guide to learn about more assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also how to assay metabolic function in live cells using your plate reader.

For reactive oxygen species, the most popular live cell ROS assay is DCFDA Assay ab113851 (green). Alternative ROS assays are available in orange (ab186028), red (ab186027), and deep red (ab186029).  ab238535 is used to measure ROS in biofluids, culture supernatants and cell lysates.

For assays designed to differentiate ROS, superoxides, and reactive nitrogen species: to assay ROS and superoxides use ab139476; to assay ROS, superoxides, and reactive nitrogen species use ab139473; to assay superoxides use ab219943.