I would like to measure ROS in Peripheral Blood Mononuclear Cells. The Oxidative Stress was induced with extracellular H2O2.
You can measure ROS by using cell-permeable dyes like CM-H2DCF-DA (DCF) available from Molecular Probes, which detect ROS. You simply incubate your PBMCs with medium containing 10 µM DCF for 30 min at 37°C and read the fluorescence intensity by flow cytometry on the FL-1 channel. The more ROS in PBMC, the higher fluorescence intensity.
Hi! DCF works well, but having compared several methods in PBMC I personally would also consider chemiluminescent methods: Specifically, lucigenin has proven extremely reliable and easy to handle (just add and measure light output in a luminometer). With lucigenin you will primarily detect superoxide anions, e.g. during the oxidative burst in your PBMC. To check your detector and the viability of your cells you may add fMLP, PMA or LPS to trigger the oxidative burst, which will occur within seconds after the stimulus. The main advantage (over fluorescent methods) is the lack of additional washing or incubation steps, which may be a source of error (e.g. if your reagents or plastic materials are not pyrogen-free).
Good luck!
Hi there,
You can use Dihydroethidium (DHE) and its works just fine.
Here is more information from Nature Protocols
http://www.nature.com/protocolexchange/protocols/414
All the best!!
ROS can be measured by DCFDA but one should be careful to remove extracellular H2O2 before addition of DCFDA to the cells since H2O2 can also oxidize DCFDA and give false positive fluorescence. Apart from flow cytometry ascommented by Carsten, ROS can also meaured using a multi-mode fluorescent microplate reader (FLUOstar Omega, BMG LabTech, USA) with temperature maintained at 37 C. Fluorescence caused by DCF in each well is measured and recorded for 30 min at 485 nm (excitation) and 530 nm (emission).
As there are some chemical pitfalls in determining ROS concentration, please be careful when selecting the dye.
Please see the following paper as an example of the problem:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3587154/
It revises one of the commonly used ROS-detecting dye - HE.
I send you my paper about evaluation of superoxide anion and hydrogen peroxide in PBMCs; however, if you wish to evaluate ROS production in human PBMCs you must use RPMI medium.
Regards
Armando
I send you my paper regarding the ROS evaluation in PBMs by chemiluminescence.
Kind Regards
Giusy
See this thorough review.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911769/
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