Integrated blood ROS generation is too time consuming, preferable could be one time point in the begin of blood ROS generation.
Dear Thomas,
Thanks for asking this very important question. You are absolutely right. PMN are the primary mediators of the rapid host defense against most bacterial and fungal pathogens. Cancer chemotherapy or reactions to cytotoxic drugs are known to cause iatrogenic neutropenias leading to severe immune deficiency. Activation of neutrophils and to enable their phagocytic properties requires increased oxygen consumption of molecular oxygen. The consumed oxygen is then utilized by NADPH oxidase generating ROS. Some of the suitable methods to quantify ROS generation are as follows:
1. Chemilunminescence amplified by luminol or isoluminol
2. the absorbance change following reduction of cytochrome c
3. The fluorescence increase upon oxidation of PHPA (p-Hydroxyphenyacetate).
I will be happy to provide you with the details of these techniques should you be interested.
Best Wishes,
Omer
Measure of of oxidative stress by CM-H(2)DCFDA through Flow Cytometry and formation of 8'-hydroxy-2'-deoxyguanosine by ELISA may be considered. Both procedures are highly robust and reproducible. In case you are interested, please have a look on our associated-publications.
Article Regulation of isocyanate-induced apoptosis, oxidative stress...
Article Impairment of Mitochondrial–Nuclear Cross Talk in Neutrophil...
There are instances where neutrophil malfunctions can be assessed through migration patterns:
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0114509
http://www.ncbi.nlm.nih.gov/pubmed/23430978
Not sure if this helps.
The main function of activated neutrophils is the generation of reactive oxygen species, purified neutrophils might artefactually generate more ROS than neutrophils in their physiologic matrix which is blood or diluted citrated blood.
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