Dear Michel,

in our lab we calibrate the BCECF signals using 10 µM nigericin (e.g.
from Fluka, Germany). Nigericin is a mycotoxin that permeabelizes the
membrane for potassium and protons. During the calibration the potassium
concentration in the bath is increased to 80 mM to set the membrane
potential to (almost) 0 mV. At this conditions, the intracellular H+
concentration will follow the extracellular H+ concentration. Wolfgang
Nett in our lab has extensively studied the BCECF calibration and
compared the results to pH measurements with pH-sensitive
microelectrodes. This is published in:
Nett, W. & Deitmer, J.W. (1996): Simultaneous measurement of
intracellular pH with BCECF and ion-sensitive microelectrodes in leech
giant glial cells. Biophys. J. 71, 394-402
We have no experience with SNAFL, but in principle it work the same way.

By the way, I did not get the original mail from Ronnie. Some other
members of the list had the same problem, is there something out of
order with the server?

Best wishes
Christian



> At 08:57 PM 11/1/1998 +0100, you wrote:
> >>>>
>
>      Dear users,
>
>      I am studying intracellular pH and would like to have some suggestion on suitable permeabilizing agent to do intracellular calibration. I have already tried digitonin at different concentration, but the respons is either no permeablization or it is so extensive so that the dye (SNAFL-2) leaks out. I am using COS-7 cells.
>
>      I am open for other suggestions.
>
>      Best regards,
>
>      Ronnie Andersson
>      ---
>      Ronnie M Andersson, PhD-student Phone:+46-8-51777340
>      Pediatric Unit Q2:09 Fax: +46-8-51777338
>      Department of Woman and Child Health Email: <mailto:[log in to unmask]>[log in to unmask]
>      Astrid Lindgren Children's Hospital
>      Karolinska Hospital
>      S-171 76 STOCKHOLM
>      SWEDEN
>
> The best way to calibrate pH is to use nigericin (5 to 10 uM), it is a K/H ionophore. Just use an intraccellular like buffer (high K and low Na)added with nigericin and adjusted to different pH.
> good luck
>
> Michel Puceat, Ph.D
> INSERM U390
> CHU Arnaud de Villeneuve
> 34295 MONTPELLIER, France
> Phone lab: (33) 4-67-41-52-44
> home: (33) 4-67-59-59-13
> Fax: (33) 4-67-41-52-42

--
Dr. Christian Lohr
Universitaet Kaiserslautern
Fachbereich Biologie
Abteilung Allgemeine Zoologie
Postfach 3049
D-67653
Kaiserslautern
Germany

http://www.uni-kl.de/FB-Biologie/AG-Deitmer/lohr.html