DAF-2 DA fluorescence

[Dr Kieran Brack]

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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hey,

this site is kinda new to me but was recommended by technical support at
Cairn Research who provide our equipment for fluorescence measurements.

I have a query that hopefully somebody might be able to help with.
We are about to submit a paper where we record DAF-2 fluorescence from the
epicaridal surface of an isolated heart, to monitor the level of nitric
oxide.

We collect excitation wavelengths of 470 / 480 / 490 / 500 nm at 535 nm
through a band pass filter of 50 nm using a bifurcated light guide (Cairn
Research).

The help we require is as follows:

After we have obtained a stable level of background autofluorescence at
the start of the experiment, we inject the dye into the solution perfusing
the heart. We continually monitor epicardial (from the surface of the
heart) fluorescence and observe a rapid and dramatic DECREASE in the
ongoing fluorescence signal.

Inititally and as with other dyes (Fura and Indo for calcium detection),
that we have used we expected a gradual increase in fluorescnence to be
seen as the dye is cleaved and begins to fluoresce in the presence of NO.

We have searched the literature and can not explain why we find a decrease
in DAF-2 fluorescence immediately after we load the dye into the isolated
heart.

We are confident we are monitoring NO as premilinary experiments have
indicated this (eg Increase in fluorescence with NO donors and decrease in
fluorescence with blockers of endogenous NO activity.

Can anybody help shed any light on this matter???

Yours truely
Kieran

[hidden email]
(University of Leicester, UK)

[Julio Vazquez]

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Kieran, 

I am not sure whether this applies to your specific situation, but we had people who used to label cells by loading them with a mitochondria fluorescent tracker. They saw the same effect: reduced fluorescence, when they expected increased fluorescence. The problem turned out to be quenching due to a dye overdose. When they reduced the concentration of the dye by 100-1000x, then things worked as expected. You could try to use a dilution series of your dye. If that doesn't solve the problem, then it's something else.

--

Julio Vazquez, PhD

Fred Hutchinson Cancer Research Center

Seattle, WA 98109-1024

[hidden email]

http://www.fhcrc.org/

On Aug 10, 2007, at 8:42 AM, Dr Kieran Brack wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hey,

this site is kinda new to me but was recommended by technical support at 

Cairn Research who provide our equipment for fluorescence measurements.

I have a query that hopefully somebody might be able to help with.

We are about to submit a paper where we record DAF-2 fluorescence from the 

epicaridal surface of an isolated heart, to monitor the level of nitric 

oxide.

We collect excitation wavelengths of 470 / 480 / 490 / 500 nm at 535 nm 

through a band pass filter of 50 nm using a bifurcated light guide (Cairn 

Research).

The help we require is as follows:

After we have obtained a stable level of background autofluorescence at 

the start of the experiment, we inject the dye into the solution perfusing 

the heart. We continually monitor epicardial (from the surface of the 

heart) fluorescence and observe a rapid and dramatic DECREASE in the 

ongoing fluorescence signal.

Inititally and as with other dyes (Fura and Indo for calcium detection), 

that we have used we expected a gradual increase in fluorescnence to be 

seen as the dye is cleaved and begins to fluoresce in the presence of NO.

We have searched the literature and can not explain why we find a decrease 

in DAF-2 fluorescence immediately after we load the dye into the isolated 

heart.

We are confident we are monitoring NO as premilinary experiments have 

indicated this (eg Increase in fluorescence with NO donors and decrease in 

fluorescence with blockers of endogenous NO activity.

Can anybody help shed any light on this matter???

Yours truely

Kieran

[hidden email]

(University of Leicester, UK)