FAQ: In Vitro ROS/RNS Assay

Q: What are the differences between the Intracellular ROS Assay and the In Vitro ROS Assay?

A: The main difference between these assays is the sample that is used.  Our Intracellular ROS Assay is performed on cultured cells.  The cell permeable DCFH-DA is added to cells and is hydrolyzed by cellular esterases to DCFH.  Once oxidized by ROS, DCFH becomes fluorescent DCF.  This assay only works on live cells or tissues because it requires esterase activity to remove the DA group.  Our In Vitro ROS Assay is typically used on cell and tissue lysates, blood, or urine.  The In Vitro assay doesn’t require active esterases and does not require the dye to permeate the cell membrane.


Q: Can this kit be used to detect ROS in bacteria?

A: In order for the Intracellular ROS assay to work, DCFH-DA must be able to penetrate live cells and then be deacetylated by cellular esterases.  Although we have not tested this kit on bacteria, it should work because DCFH-DA is cell permeable, and there are publications showing the use of DCFH-DA with bacteria.


Q: Will this kit work on yeast or fungal organisms that have thick cell walls?

A: Although we have not tested this assay on yeast cells, a literature search reveals that other researchers are able to successfully use the DCFH dye on yeast cells.  This assay requires the cells to be lysed prior to testing.


Q: Is this kit compatible with tissue samples?

A: The In Vitro ROS/RNS Assay is suitable for use with tissue samples to measure ROS as long as you have access to a fluorescent plate reader.  The product manual contains sample preparation guidelines for tissue lysates, which is to homogenize or sonicate tissues in 1X PBS.  Due to the transient nature of ROS, we recommend using freshly prepared samples, however frozen tissues can be used if frozen for no longer than 2 months at -80ºC. 


Q: Can this kit be used on cells that have been fixed in paraformaldehyde

A: Our In Vitro ROS/RNS Assay kit is suitable for cell or tissue lysates and does not need to be performed on living cells.  ROS/RNS has a very short half life already and is likely to be more unstable if the cells are fixed.   For this reason, we do not recommend this assay with fixed cells.


Q: Do you have a positive and negative control for these assays?

A: Most researchers using our ROS Assay kits are measuring the relative fluorescence of treated vs. untreated samples, with the treated samples being exposed to either a stimulator or an inhibitor of ROS. The untreated cells would therefore be considered the baseline control, even though it’s technically not a true “negative” control. The results of the treatment are reported as a fold-change compared to the untreated samples.


Q: Can this assay be used to measure ROS levels in frozen samples?

A: The In Vitro ROS/RNS assay can be used on samples frozen for no longer than 1-2 months at -80ºC.  We don’t recommend storing samples for this long, if possible, since the best results will come from fresh samples.   For samples that have been frozen longer than this, best results will be obtained by testing a stable marker of oxidative damage such as Protein Carbonyl, 8-OHdG or 4-HNE.


Q: Will ROS be generated during the sample preparation steps, such as trypsinisation?

A: The In Vitro ROS/RNS assay detects the total free radical presence of a sample, which includes hydrogen peroxide as well as other free radicals including the ones shown in the product manual.  ROS is produced outside of oxidative stress and can be induced during sample preparation.  Our ROS assays do not differentiate between the source of ROS production, but an increase in ROS/damage levels is expected to be uniform if induced by sample preparation stress.  ROS levels due to sample preparation are not expected to be significant and can be considered background across all samples.  If there is concern about subjecting samples to additional ROS, consider using our Intracelluar ROS assay which measures ROS in cultured cells, eliminating the additional sample preparation steps.


Q: How much sample should I use?

A: We don’t provide a recommendation on sample dilution because the assay is dependent upon the ROS level in sample, rather than protein concentration, and this is influenced by sample type, preparation techniques, and storage condition.  We recommend performing a sample titration against the standard curve prior to running the experiment to determine if a dilution is necessary so that the sample values will fall within the middle of the standard curve.  For example, you could try undiluted, 1:2, 1:10 and 1:20 dilutions.  Despite the dilution used, the sample volume added to the well should remain 50 µL.


Q: What is the minimum volume of urine that I can detect with this kit?

A: It is difficult for us to provide a minimum volume requirement for this assay because the ROS level is sample dependent and is influenced by sample preparation techniques and storage conditions.  We recommend performing a sample titration against the standard curve prior to running the experiment to determine the minimum volume necessary that will still fall within the middle of the standard curve.

You may also want to consider using the 8-OHdG ELISA kit #STA-320 because 8-OHdG is very stable and easy to detect in urine.


Q: How do you detect the different ROS/RNS species that are shown in the manual, such as peroxyl radical, peroxynitrite, and nitric oxide?

A: The In Vitro ROS/RNS assay detects the total free radical presence of a sample, which includes hydrogen peroxideas well as the other free radicals shown in the product manual.  Hydrogen peroxide is the predominant form and there is no way to exclude the other free radicals or to isolate just one specifically.  The graphs in the product manual were generated using each compound specifically in the assay to show that they are detectable; however it is not possible to distinguish them from within a sample.  Because the majority of ROS in a sample is from hydrogen peroxide, this can be measured against a hydrogen peroxide standard curve.  The DCF standard is used to get a general measurement of all free radicals in a sample and the hydrogen peroxide standard is used to measure the hydrogen peroxide levels in a sample.  It is recommended to run the hydrogen peroxide standard curve to confirm that the assay is working properly.


Q: Can I use the DCF standard curve to analyze my results?

A: The DCF standard curve is optional and is used to ensure that the plate reader is working properly and that the dye can be detected at various concentrations; however the DCF standards do not go through the assay protocol and cannot give any indication of free radicals in a sample.  The hydrogen peroxide standard curve is more useful because it is used to measure the hydrogen peroxide levels in the sample, which is the predominant form of ROS and will be the majority of the signal detected in the sample. 

It is fine to use the DCF standard curve if you are interested in presenting your results as relative comparisons between samples.   The DCF standard curve does not give any indication of free radicals in a sample and a DCF value is not meaningful, so it is probably not a good idea to present your result values as DCF (nM).  If you have to present your results as absolute values, you should use the hydrogen peroxide standard curve, which will reflect the amount of hydrogen peroxide in the samples.


Q: Is the catalyst and DCF added to all wells, including both standards?

A: No, if using the DCF standard it is not necessary to add either the catalyst or DCFH solution to those wells.  Once the DCF standard is prepared, it is complete and can be read immediately.  The catalyst and DCFH solution should be added to the wells containing the H2O2 standard and all the wells with your samples.