DCFDA / H2DCFDA - Cellular ROS Assay Kit (ab113851)

In the protocol we recommend to run the assay in the absence of phenol red as it can increase the background. The background seems to be more of a problem on spectrophotometers than on flow cytometers.

Because it is well known that phenol red gives reading at 530nm we do not have development data using this kit in the presence of phenol red.

I would suggest starting with 50 µM of TBHP as a positive control as we have observed linearity of signal when seeding Jurkat cells at 200,000 cells per well with treatments of TBHP from 50 – 500uM.

The amount of TBHP to use will however depend on the sensitivity of the cell line to TBHP (HL60 and Jurkat cells are very sensitive whereas HepG2 are very insensitive). Therefore you may have to optimise this concentration depending on your cells sensitivity to TBHP.

The vehicle control or vehicle titration refers to the diluent used in the test compounds. Some researchers dilute their compounds in ethanol or DMSO. If the test compounds are assayed in titration, we suggest for the vehicle or diluent of the test compound to be also assayed in titration. The vehicle/diluent is simply a negative control. It simply shows that it is only the compound TBHP that is responsible for the increase in ROS and not some other confounding variable such as the diluent itself

In this case ter-Buytyl hydroperoxide solution is made up in 5M decane. 5mM decane could be used to treat the control culture, as the TBPH is given at 1000X concentration.

A measurement at 570nm will not detect the dye (DCFDA). The dye has the emission maximum at 529nm. We give 535nm, as the old plate reader with UV lamp can measure this wavelength and this works with the kit. I therefore propose that you buy the kit only, if you find a plate reader capable of reading at 535 or 529nm.

The lab says fixation will cause the dyes to leak from the cells. I think the assumption is that fixation may damage cell membranes. I did find a paper which describes fixation using methanol or acetic acid, after staining with a DCFDA derivative. DCFDA is the reagent that stains ROS in the kit ab113851, and I suspect that it is the green dye in the other two kits, the ROS/NOS and superoxide assays, though our source for those kits has not confirmed. A modified fixed staining method for the simultaneous measurement of reactive oxygen species and oxidative responses. Shen et al, 2013. PMID: 23178299 http://www.ncbi.nlm.nih.gov/pubmed/23178299

The lab has informed me that the cells must be alive, so fixed cells will not work with this kit.

In our experience, running the flow cytometer with cells in media did not appreciably affect the data – there was marginally less “background” if the cells were washed, but the slight improvement in data signal did not warrant the extra steps. Hence we recommend the simpler procedure of reading directly in media.

Staining of the cells before treatment allows (1) bulk labeling of the cells with DCFDA (2) the compounds/treatments to remain on the cells at the time of the reading. For example (with drug treatments), if treatments were done first, then subsequent addition of DCFDA would lead to a dilution of the treatments (unless drugs were also spiked into the DCFDA solution). Again, this is a case of protocol simplification. Also note that for some drug treatments the effect on ROS levels can be rapidly reversed e.g. a drug can induce ROS levels in a cell, but cellular ROS levels can decrease rapidly as soon as the drug is removed/diluted.

1) The plate should be read with buffer (100uL/well). Cells must be alive during the reading.

2) For a plate reader assay, it is preferable not to use phenol red as there could be background. If you need to read to phenol red, make sure to include appropriate negative controls = (1) unstained treated cells with media, (2) media only with no cells."


3) It is essential to read in the presence of compounds. If you need to read in media ensure that untreated controls are also read in media and as in answer #2 make sure you include appropriate negative controls to take into account any background signal.

Some types of tissue cultue cells adhere only weakly to plastic or glass and are easily washed away with buffer washes as you describe. Some things to try:

1. Make sure to was the cells very gently. Use a multi-channel pipettor to gently add was buffer by letting it slowly stream down the side of each well.

2. Alternatively, you can coat the plates to increase cell adhernce. Poly-L-lysine is often used to coat wells for this purpose. Alternative treatments for coating the plastic wells may exist which are better for your application however.

The primary difference between white and black plates is their reflective properties. White plates reflect light and will maximize light output signal, black plates absorb light and reduce background and crosstalk. For this reason, white plates are commonly used for luminescent assays and black plates are used for fluorescent assays. For this reason we recommend black plates with this kit. Thus white plates may result in higher crosstalk and higher background with this kit.

Regarding second question, if toxicity assay is not of interest, just add 100 uL/well of 1Xsupplemented buffer solution after the washing step.