Re: ROS detection **vendor reply**

[Kilgore, Jason A.]

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** Vendor Reply **

Sometimes what can happen, particularly when overloading a cell (too high concentration, or too long of loading time) is that dye-dye quenching can occur.  This has been seen with various fluorescein derivatives.  Upon photobleaching, the FRET efficiency that causes the dye-dye quenching is reduced, leading to an unquenching of the dyes and an increase in cellular fluorescence.  At some point the FRET efficiency is reduced to the point that there is no longer significant dye-dye quenching, and at that point photobleaching effects become apparent.

I recommend you try a range of loading concentrations downward.  If what I say is true, then you'll see that, as you decrease the concentration, the initial intensity will increase, plateau, then decrease.  It's at that plateau where you will want to load your cells.

Cheers,

Jason


Jason A. Kilgore
Technical Application Scientist
Molecular Probes Tech Support
Life Sciences Solutions

Thermo Fisher Scientific
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Stefano Giovannardi
Sent: Thursday, November 06, 2014 2:32 AM
To: [hidden email]
Subject: ROS detection

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Dear all,

I am trying to monitor ROS production by different treatments on different cell lines using DCFH-DA (dichlorofluorescein diacetate), the method is quite well established and is commonly used with plate readers and FACS (http://www.ncbi.nlm.nih.gov/pubmed/10490282) but...

looking at the loaded cells in wide field fluorescence I start with very dim fluorescence levels that increase, over time, by more than 10 times; in 15-30 seconds the cells reach a sort of plateau and then photobleaching takes over and the fluorescence returns slowly to low levels. Different cell lines behave slightly differently in the timing of the process.

I was thinking about a sort of photo activation process but literature does not help too much.
Of course in these conditions is impossible to establish a correlation between ROS production and fluorescence levels.

Anybody experienced this phenomenon? Any suggestion?

Thanks, best regards
Stefano




Stefano Giovannardi PhD
Cell Physiology Lab
Dipartimento di Scienze Teoriche e Applicate Universita' dell'Insubria via A. da Giussano, 12
21052 Busto Arsizio (VA), Italy

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