Rhodamine 123 is a distributional membrane potential probe. The dyes distribute
according to the nernst equation, but once inside the cell they find an
intracellular binding site they hang on to acting as an intracellular sink.
This allows more fluorochrome to enter the cell, leading to a substantial
signal enhancement. As nicely described in Howard Shapiros book, in mamalian
cells it can be used at high concentrations to act as mitochondrial MP
measurement. When the dye is washed away the dye is retained in the
mitochondria as it is in distributional balance with the remaining dye in the
cells cytoplasm. However, if you read Howards book you will find better ways to
do that, (especially in his recent work on distributional MP staining) and also
how it all works. Remember that the signals are dependent on the MP, and the
number of sinks (e.g. volume of the cell and concentration of binding sites)
In bacteria the problem is that most of them have efflux systems for small
cationic lipophilic molecules. Ethidium Bromide and RH123 are effected by those
pumps, thus do not label pumping cells. Dyes with higher influx kinetics or
used at very high extracellular concentrations can overcome the efflux pumps.
Propidium Iodide (PI) is so far the most reliable dye exclusion stain, e.g. it
does not go in intact (potentially reproductive growing) cells. If you mix a
heatfixed and a hapy non-pumping lot of cells and stain them with PI you can
counterstain with different concentrations of RH123 to see at what
concentrations only the intact cells pick up the RH123 and when both
populations become green fluorescent. I got the pictures in my tutorial if you
need them
Good luck
Gerhard
-----Original Message-----
From: Surapon Worapongpaiboon [SMTP:raswp@mahidol.ac.th]
Sent: Tuesday, September 14, 1999 3:00 AM
To: Cytometry Mailing List
Subject: Rhodamine VS Propidium iodide
Dear all: Does anyone know if rhodamine stain dead cells or just
viable cells? and is it true that dead cells don't pick up propidium iodide? I
just want to use only rhodamine as a single dye to exclude dead cells and also
an indicator of dye efflux. Thanks Surapon Worapongpaiboon, M.D.
Department of Pathology Ramathibodi Hospital Mahidol University Bangkok,
Thailand
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