DCFDA / H2DCFDA - Cellular ROS Assay Kit(AB113851)

Effect of anethole on excessive ROS generation in hMSCs. hMSCs were exposed at 2 mM H2O2 for 30 min and incubated for 2 days in presence or absence of 50 μM anethole. ROS was measured by staining the cells with DCFDA cellular ROS detection assay kit according to the manufacturer's instructions. ROS generation was observed under a fluorescence microscope at 200× magnification.

Image from Rhee YH et al., BMC Cell Biol, 2018, 19: 12, Fig. 3B.; doi: 10.1186/s12860-018-0163-2 Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/

DCFDA ROS assay used to study Doxorubicin cardiotoxicity.

Kobashigawa et al. (Pubmed 25127116) used the DCFDA ROS assay ab113851 to investigate the causes of the protective effects of metformin (Met) treatment in Doxorubicin (Dox) induced cardiotoxicity.

They identified that in metformin treated H9c2 rat immortalized cardiomyoblasts, Met treatment reduced ROS levels induced by Dox (A). Values represent mean ± S.D. (n = 4).

In combination with other assays, they developed the hypothesis that Dox induces increased ROS expression, leading to increased calcium levels and cell death, and that Met reduces this effect by increasing AMPK expression.

Kobashigawa et al. (Pubmed 28056084)

p38 MAPK pathway involved in oxidative injury to HCECs challenged with C. albicans.

p38 MAPK pathway involved in oxidative injury to HCECs challenged with C. albicans. Increased ROS generation in HCECs challenged with C. albicans and inhibition by the p38 activation inhibitor SB203580.

Image from Hua X et al., Sci Rep, 2017, 7: 10421, Fig. 4A.; doi: 10.1038/s41598-017-09636-w Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

Jurkat cells were labeled with DCFDA (20 μM) or unlabeled (none) and then cultured an additional 3 hours with or without 50 μM tert-butyl hydrogen peroxide (TBHP) according to the protocol. Cells were then analyzed on a fluorescent plate reader. Mean +/- standard deviation is plotted for 4 replicates from each condition. TBHP mimics ROS activity to oxidize DCFDA to fluorescent DCF.

Acute Effect of anthracyclines on ROS production in HL60 cells.

Labeled HL60 cells were treated with idarubicin or doxorubicin for 4 hours at multiple doses according to the protocol. At the end of the treatment cells were read end point in a fluorescent plate reader (Perking Elmer-Wallac 1420 Victor 2 Multilabel plate reader). Mean +/- standard deviation is plotted for 3 replicates from each condition. The dotted line represents the mean of 24 replicates of HL60 cells treated with 0.5% DMSO.

Reactive oxygen species (ROS) measured using the DCFDA assay in human primary articular chondrocytes. Cells were treated with 100 μM tert-butyl-hydroperoxide (tBHP) alone (4 h) ± pre-treatment with apigenin.

Image from Davidson et al., Sci Rep, 2018. Fig. 2B.; doi: 10.1038/s41598-018-35455-8 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

ab113851 (DCFDA) labeled and unlabeled Jurkat cells were treated with 50 μM tert-butyl Hydrogen Peroxide (tbHP), then analyzed by flow cytomet

ROS levels analysis in human immortalized cardiomyocytes cells with ab113851 under different treatment conditions.

Data are mean ± SEM of three different experiments. H2O2 800μM was used as Positive Control. The values of fluorescence intensity at each time point are indicated as the ratio of the value at specific t-time point on the value at time point zero (first measurement) (t-time point/t0). Briefly, cells were plated (seeding density 2.5 x 104 cells/cm2) in FBS-supplemented medium w/o phenol red onto a 96 black well plate. After 24 hours the cells were washed one time with 1X buffer (provided in the kit), then the cells were incubated with DFCDA 10 μM for 30 min at 37° C protected from light. Following incubation the wells were washed with PBS and the cells were exposed to treatments of interest in FBS-supplemented medium w/o phenol red. ROS production was determined immediately by measuring the formation of fluorescent dichloro fluorescein (DCF), using a PerkinElmer VICTOR3, at an Ex-485 and Em-535 nm. Measurements were done every 30 min for six hours. The value of fluorescence intensity at each time point is reported. The value reported was obtained by the ratio of fluorescence at specific time point on fluorescence at time 0, which was measured immediately after DCFDA incubation.