Re: Transmembrane potential and Antibody staining...
Howard Shapiro (hms@shapirolab.com)
Mon, 14 Apr 1997 09:13:31 -0400 (EDT)
The spectrum of DiOC6(3) is pretty close to that of FITC. However, what
everybody seems to forget about membrane potential staining with cyanine
dyes is that cyanine dyes with the same side chain length behave pretty much
identically (although the thiacyanines tend to be more toxic and less stable).
If you incubate cells with 100 nM DiOC6(3), the concentration in the cells
will be something like 100 uM, because both the membrane potential and
hydrophobicity drive dyes into the cell, making the concentration ratio
inside/outside higher than the Nernst equation would predict on the basis of
membrane potential alone. A lymphocyte with a 200 fl volume will therefore
contain about 12 million dye molecules, most of which will be able to
fluoresce with about the same quantum efficiency one would expect from an
antibody label. There will be some spectral shifting of DiOC6(3) toward the
red at this concentration, the result being that it won't be easy to detect
a few tens of thousands of antibody molecules labeled with a dye emitting at
a longer wavelength.
The simplest solution to this problem is to use a different cyanine dye
membrane potential probe, specifically (assuming you want to use single
laser excitation at 488 nm), DiIC1(3), or hexamethylindocarbocyanine iodide
(available from Molecular Probes). This dye (the same chromophore as Cy3)
is less hydrophobic than DiOC6(3) because it has methyl rather than hexyl
side chains, so you'll have fewer dye molecules in the cell. The absorption
maximum is at 539 nm, but the million or so molecules you're looking at will
give you an acceptable signal with 488 nm excitation; emission maximum is at
564 nm, but you can detect it adequately through a filter designed for PE.
You can then use fluorescein antibodies; since DiIC1(3) has essentially no
emission in the green, interference will be minimal.
If you happen to have a system with 488 and red (633 nm He-Ne or 635 nm
diode) excitation, you can also use DiIC1(5) (hexamethylindodicarbocyanine
iodide) as the membrane potential probe; this is the same chromophore as
Cy5, with maximal excitation at 641 nm and maximal emission about 660-670
nm. Laser grade DiIC1(5) (99% pure) is available from Acros, which is a
division of Fisher Scientific; they acquired the product line of Eastman
Organic Chemicals from Kodak a few years ago. Acros should also have
DiOC6(3) and DiIC1(3), possibly at a lower price than Molecular Probes.
More information on membrane potential staining can be found in Practical
Flow Cytometry, in my article in Methods in Cell Biology, Volume 41, in a
forthcoming article in Current Protocols in Cytometry, and in my U. S.
Patent 4,343,782, which (with related patents in 12 other countries) covers
every use of membrane potential stains on cells other than nerve and muscle
cells; commercial users take note.
-Howard
CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
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