I am using luminol for ROS determination in Neutrophils. first of all i have problem in dissolution of Luminol. I made a stock of 3M in DMSO. Briefly I use 100 microleter of neutrophils from 5 million per ml with 100 microleter of HBSS and 1 ul of luminol (10mM). After incubation for 10 minutes at 37 C, PMA was added and acquired.
1. There are intraday variations between the control samples. what is the possible reason for that (all steps are same?
2 luminol is also not that much soluble even in room temperature.
Hello Amit,
There are a variety of limitations using luminol to measure "oxidative stress". To begin it can react with molecular oxygen as well as light under various conditions and this may lead to intraday variations as well as make analysis difficult. See the following articles: Article Measuring reactive oxygen and nitrogen species with fluoresc...
http://circres.ahajournals.org/content/circresaha/89/3/224.full.pdf
Luminol will also most likely detect peroxynitrite with nitric oxide from iNOS in neutrophils reacts with the superoxide being produced.
I would recommend looking into other methods in the papers above as well and maybe even combine a few techniques to accurately measure ROS from neutrophils.
Best,
Hi Amit,
Luminol is a very poor detector of ROS at physiological pH. Have you considered using Pholasin. You can also measure ROS from neutrophils in diluted blood where they will be in contact with all the other cells. The neutrophils behave differently when isolated than in contact with other leucocytes. We would be pleased to help you further. You can also download a lot information from the website www.knightscientific.com
All the best
Jan
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