Hi there,
I want to generate NADPH oxidase (NOX)-specific induction of ROS in vitro and was wanting to try and avoid using H2O2 since this is a downstream product and ROS is naturally generated in cells.
I'm struggling to find compounds/cytokines in the literature that specifically induce NOX. I plan to investigate the effects of oxidative stress on regulation of the avian HPA-axis feedback mechanisms.
Thanks in advance.
NADPH oxidase is activated by many agonists including angiotensin II, PDGF, TNF, alpha 1 agonists and multiple others depending on the NADPH oxidase isozyme and the cell type. One of the most classical and direct ways to induce NADPH oxidase 2 (Nox2) is to use phorbol ester, an activator of protein kinase C. However, I don't think we know whether there is any one agonist that only induces NADPH oxidase. I could be wrong. Most if not all stimulants effect activation of more than one signaling pathway, at least in mammalian cells. I do not know whether the same holds true for avian cells. Perhaps the best way, albeit artificial, would be to identify the Nox isoform and its subunits in your cells and overexpress a key subunit using a plasmid or viral vector.
Dear Dr Walker,
Not clear from your Q, but this depends a lot on what kind of in vitro system you are using. For example, if vascular cells (VSMC or EC) ANG II would be a good stimuli, whereas if you are looking at inflammatory/immune cells (macrophages or monocytes) classical activation by e.g. LPS works great. Agree with Dr Pagano that induction of Nox2 by using phorbol ester is an option. //Mattias Carlstrom
I agree with Dr Pagano and Dr Carlstrom, Moreover, I suggest that the chronic ethanol treatment of microglia and astrocytes induces NOX activation.
Want to second use of LPS or PMA for macrophages as mentioned by Patrick and Mattias. If you choose to use LPS, it will require a pre-stimulation step of few to many hours. With regards to PMA, aliquot it in single to use volumes.