I am trying to measure PMN intra-cellular ROS using DCF-DA by microplate reader. Sometimes, the fluorescence intensity of blank (only meida+DCF-DA) is higher than that of sample readings. Can anybody explain?
Monirul,
The DCF assay generates a lot of artefarct and it is not recomended.
Free Radic Biol Med. 2012 Jan 1;52(1):1-6.
Please read recent publications listed below and let me know if you have any questions reagrding the better assays.
1) Measurement of Reactive Oxygen Species...A Scientific Statement From the American Heart Association. Circ Res. 2016 Aug 19;119(5):e39-75.
2) Methods for detection of mitochondrial and cellular reactive oxygen species.Antioxid Redox Signal. 2014 Jan 10;20(2):372-82.
These papers are available at my Reserach Gate page.
Sergey.
Hi Monirul,
Unfortunately, there is no perfect probe to accurately and specifically measure intracellular ROS production (as you can find in Dr. Dikalov's recommended FRBM paper, and some others, too). We had much better success employing DRH123 as oppose to DCF in experiments with endothelial cells and PMN. Also, you may consider using O2- "specific" probe, L-012 (chemiluminescence probe), to chase PMN oxidant production. (Am J Physiol Heart Circ Physiol. 2009 Sep;297(3):H920-9.) (Free Radic Biol Med. 2014 May;70:167-73.) Keep in mind that PMNs produce ROS very quickly, so don't miss the time window...
Finally, be extra careful in choosing the microplates (e.g. plastic) for fluorescence measurement. The results obtained from a "microplate reader" differ from the "fluorescence spectrometry/glass cuvettes" obtained ones, withe the latter being more sensitive and more accurate. To get a good signal using microplate approach you have to induce a massive oxidative stress to the cells!
Best of luck
Gedas
Md. Monirul Islam Hi can you please share the protocol for the assay with microplate reader using DCHF dye? Did you manage to optimize it for your analysis?
Also, do you use black microplates with transparent bottom or transparent cell binding microplates?
Faria Khan I used transparent cell binding 6 well plate.
First, I treated the cell with a compound. Then, after desired incubation time, I washed the cell and added 10 micro-molar DCF-DA for 30 min and again washed the cell. After that, the culture dishes were replaced with phenol red free fresh RPMI-1640 and fluorescence was measured by micro-plate reader. Media with DCF-DA was used as blank.
Md. Monirul Islam Thanks for getting back to me and for the protocol. I was wondering did using transparent plates cause a difference in the reading?
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