saline blue

[mmodel]

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Has anyone tried saline blue to measure intracellular chloride?
Thanks!

Mike Model

[George McNamara]

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Hi Mike,

I believe SPQ and/or MQAE was the standard of care in small molecule
Chloride ion sensors back in my MetaFluor - Image-1/FL support days (but
UV excitation if I recall correctly). See, for example (though
two-photon excitation), Kovalchuk 2012, PubMed 22753606:
This protocol describes a technique for high-resolution chloride imaging
of living cells using a quinoline-based chloride (Cl(-)) indicator dye,
MQAE(N-[6-methoxyquinolyl] acetoethyl ester). Bath-applied to acute
brain slices, MQAE provides high-quality labeling of neuronal cells and
their processes. In living anesthetized mice, cortical cells are labeled
using the multicell bolus loading procedure. In combination with
two-photon microscopy, this procedure enables in vivo visualization of
cell bodies of neurons and astrocytes as well as some astrocytic
processes and allows one to monitor changes in the intracellular
chloride concentration in dozens of individual cells.

See also Munkonge 2004, PubMed 15463953 - cystic fibrosis studies have
driven a lot of the Cl- ion imaging.
The use of the halide-sensitive fluorescent probes
(6-methoxy-N-(-sulphopropyl)quinolinium (SPQ) and
N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE)) to measure
chloride transport in cells has now been established as an alternative
to the halide-selective electrode technique, radioisotope efflux assays
and patch-clamp electrophysiology. We report here procedures for the
assessment of halide efflux, using SPQ/MQAE halide-sensitive fluorescent
indicators, from both adherent cultured epithelial cells and freshly
obtained primary human airway epithelial cells. The procedure describes
the calculation of efflux rate constants using experimentally derived
SPQ/MQAE fluorescence intensities and empirically derived Stern-Volmer
calibration constants. These fluorescence methods permit the
quantitative analysis of CFTR function.

FPs:
Clomeleon FP sensor (ratiometric YFP:CFP more useful for halides than
straight up EYFP) has some publications. A 2008 paper report, Markova
2008, PubMed 18279971, reported improvement. Might be even better if
tethered (fusion protein) to the ion channel(s).
PubMed search:    chloride ion fluorescent protein biosensor
turned up Watts 2012, PubMed 22506078, on mbYFPQS (membrane bound by
palmitylation instead of fusion to the ion channel).


George
p.s. also useful to multimerize FP biosensors (as long as doing so does
not mess up function), since, for example, 100 FP's in one voxel, is
more useful than one FP in each of 100 voxels. See also
http://works.bepress.com/gmcnamara/42 for more (and other pages on
Tattletales on my bepress site).


On 8/12/2015 10:51 AM, MODEL, MICHAEL wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Has anyone tried saline blue to measure intracellular chloride?
> Thanks!
>
> Mike Model
>
>    

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/42