Fast And Efficient Peroxynitrite Quantification In Intact Cells

October 05, 2016

scuola superiore sant'anna
life science institute
post-doc grant researcher

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Company:

AAT Bioquest

Product Name:

Cell Meter™ Fluorimetric Intracellular Peroxynitrite Assay Kit *Optimized for Flow Cytometry*

Catalog Number:

16317

We found this kit as we were looking for a way to measure peroxynitrite production in endothelial cells exposed to oxidative stress, as per a reviewer's request. this kit was our choice as it was optimized for flow cytometry therefore allowing fast and effective quantification of fluorescent intensity.

Experimental Design and Results Summary

Application

Flow cytometry

Starting Material

Porcine aortic endothelial cells, exposed to oxidative stress-inducing treatment

Protocol Overview

Cells were treated with PMA for 48h to induce oxidative stress; therefore following manufactorer's instructions, cells (5x10^5/ml) were resuspended in fresh complete medium, and incubated with DAX-J2 PON probe, for 1 h at 37 °C. Finally, cell fluorescence was quantified by flow cytometry using a FACScalibur.

Tips

Always include a negative control (w/o probe) to tune out cellular autofluorescence.

Results Summary

We were able to demonstrate that PMA treatment induced an increase in peroxynitrite production, and that our treatment (I.E. silencing of target genes) blocked wuch increase; these effects were quantified as shifts of the green fluorescence peak in the FLH-1 histogram toward the right (increase) or left (decrease) end of the diagram. therefore the kit is a good tool if you want to investigate the effect of some treatment on peroxynitrite intracellular levels.

Additional Notes

None

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Summary

The Good

Fast, little need for protocol optimization, perfect if you're in a hurry

The Bad

Works only for flow cytometry, so if you need to confirm your data by fluorescence microscopy, you need the specific kit

The Bottom Line

This kit is a perfect solution to measure this specific kind of oxidative stress byproduct, particularly useful if you're interested in endothelial cell physiology

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