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: 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 your Intracellular ROS Assay be used with a microscope to detect ROS?
A: This assay detects ROS in cultured cells and can be read with either a fluorescence microscope or a fluorescent plate reader. It is not necessary to prepare a lysate if cells are cultured on a black plate.
Q: How do I treat my cells?
A: It is important that the DCFH is added to the cells before applying a treatment so that it is available to immediately bind to any ROS that is producedand capture all changes resulting from ROS. Once inside the cells, DCFH-DA is hydrolyzed by cellular esterases to DCFH, which gets trapped inside the cells. Upon oxidation by ROS, the non-fluorescent DCFH is converted to fluorescent DCF. Because endogenous ROS is continuously produced by cells, you should treat your cells with your drug within one hour after adding DCFH-DA dye to the cells. When performing a drug treatment, it is recommended to culture the cells in a black plate so that you can perform a kinetic analysis to monitor the drug effect over the incubation period. If you culture cells in a clear plate, you will need to transfer to a black plate for fluorescence reading and then you will only have one data point per sample.
Q: What is the recommended protocol for hydrogen peroxide treatment of cultured cells?
A: To induce ROS production, we treat HeLa cells with 100 µM-1 mM hydrogen peroxide for 1 hr, but different cells may require a different dose or treatment time and some cells may show toxicity if the concentration is too high.
Q: Can I add treatment and let it incubate for 24 hours?
A: The assay will work past the 1 hour incubation time stated in the protocol, but the results will depend on the specific treatment you are using. For this type of experiment, we recommend seeding your cells in a black well plate so that you can perform a kinetic analysis on your cells to determine the optimal time of your treatment. It is important that the DCFH is added to the cells before you apply your treatment in order to capture all changes resulting from ROS.
Q: My drug treatment is 72 hours; will the DCFH-DA be stable for that long? Will it leak out of the cells?
A: Although we have not tested this assay for 72 hours, it should be fine because DCFH is very stable. If it does start to degrade over the long incubation period, you may see a decrease in the fluorescence readings, however this should be a uniform degradation and you will still be able to obtain relative comparisons between samples. You may want to consider culturing the cells in a black plate so that you can perform a kinetic analysis to monitor the drug effect over the incubation period. If you culture cells in a clear plate, you will need to transfer to a black plate for fluorescence reading and then you will only have one data point per sample.
Q: Will this assay work with any cell type?
A: Yes, the Intracellular ROS Assay (STA-342) will detect ROS independent of cell type.
Q: Can I use this with suspension cells?
A: Yes, the Intracellular ROS assay kit (STA-342) can be used for both adherent cells and cells in suspension culture. For suspension culture, the labeling method is same: cells are incubated with 1X DCFH-DA for 30-60 min at 37C and the excess dye is removed by washing cells with DPBS. To wash suspension cells, spin down cells and wash the pellet with DPBS.
Q: If my cells are methanol sensitive, do I need to increase the volume of the 0.1X diluted dye?
A: For cells that are sensitive to methanol, we recommend making further dilutions of the dye to 0.1X or 0.01X final. The volume of the DCFH-DA dilutions used in the assay will not need to be altered. The sensitivity is not affected with the 0.1X dilution, which can still be read with the fluorescent plate reader; however the 0.01X dilution will be too dilute to be detected with the plate reader and will need to be read with FACS.
Q: Is the reaction of DCFH to DCF reversible?
A: Once the DCFH reacts with ROS/RNS, it becomes DCF, which is stable. The reaction is not reversible.
Q: How do ROS levels correlate to DCF fluorescence?
A: The DCF fluorescence is directly proportional to ROS levels, however since this kit does not use an ROS standard curve, it is not possible to correlate RFU to a specific ROS value. The standard curve is prepared to confirm that the assay is working, rather than for quantifying results and results are typically presented as RFU values or as relative differences between samples (Figure 3 on page 6 of the product manual).
Q: Can I fix cells and do timepoint measurements with a fluorescent microscope?
A: We have not tested this assay on fixed cells and don’t know how it will affect the assay. We don’t know if fixation will introduce ROS that will oxidize the probe, or if ROS will no longer be produced in the cell. If you are using an inverted microscope, it is not necessary to fix the cells because the wells can be visualized directly under the microscope at various time points without stopping the reaction.
Q: Why am I getting a high background when I view under the microscope?
A: The dye used in this assay is light sensitive and will auto-oxidize when exposed to light, which will increase the fluorescence. If you are experiencing high background, an option is to remove the buffer and replace with fresh buffer. If viewing under the microscope with the light on, it is best to view quickly to avoid the fluorescence increase.