Contents

Store kit at 4°C in the dark immediately on receipt. For longer term storage of 12 months, store kit at -20°C.

Parameters 

Fluorescence is measured at Ex/Em = 485/535 nm

Description 

Dichlorodihydrofluorescein-diacetate (DCFH-DA) is a fluorogenic dye that measures hydroxyl, peroxyl and other reactive oxygen species (ROS) activity within the cell. After diffusion in to the cell, the acetyl groups on DCFH-DA are cleaved by intracellular esterase to yield the non-fluorescent compound (DCFH) which is rapidly oxidized to highly fluorescent 2',7'-Dichlorodihydrofluorescein (DCF) by ROS. DCF can be detected by fluorescence spectroscopy with maximum excitation and emission spectra of 504 nm and 529 nm, respectively. The fluorescence intensity is proportional to the ROS levels within the cell cytosol. The ROS-inducing drug Rosup is used as a positive control. Each reactive oxygen species assay kit contains sufficient materials for approximately 1000 measurements in 96-well-plate format.

Notes 

1. Rosup is usually adopted at a concentration of 100μM (given different cell types, the recommended concentration is 100-400 μM). In general, a significant increase in ROS can be observed 30 min-4 h after stimulation. The effect of Rosup may vary greatly among different cell types. If the increase of ROS is not observed within 30 min after stimulation, the induction time can be prolonged or the concentration of Rosup can be appropriately increased. If ROS increase too quickly, the induction time can be shortened or the concentration of Rosup can be reduced appropriately. 

2. If the negative control groups without stimulation have strong fluorescence, dilute DCFH-DA in accordance with 1:200-1:500 to set the final concentration of DCFH-DA at 2-5μM. The incubation time can also be adjusted within 15-60 min based on the situation. 

3. Rosup is only applied in positive control samples, and it is not necessary to add Rosup in every sample. 

4. After DCFH-DA incubation, clean the remaining probes that have not entered the cells which may cause a high noise.