Good point on the NMR, Following Satre and Klein, I tried this, you just need a thin tube put into the NMR tube to bubble the air with live cells in the tube, and to carefully adjust the airflow so that the the cells don't bubble out into the NMR machine.... you also get the pH of vesicles/ mitochondria, and levels of IP6, nucleotide triphosphates and diphosphates. Its fun, cheap, easy, and quick.
just pubmed
dictyostelium NMR pH 31P
and you can see various improvements, such as using methyl phosphonate as a probe
Richard Gomer
________________________________________
From: DICTY [[log in to unmask]] on behalf of Rob Kay [[log in to unmask]]
Sent: Tuesday, October 8, 2019 10:52 AM
To: [log in to unmask]
Subject: Re: [DICTY] pH-sensitive dyes
Dear Marianne,
The definitive way of measuring intra-cellular pH is by 31P-NMR as practiced many years ago by Gerard Klein and Michel Satre and also ourselves (J Cell Sci 83, 165 1986). The cytosolic pH by this method is about 7.3.
But of course you need a specially adapted NMR machine with air bubbled to your cells and rather a lot of cells, and you cannot pick up fast responses (at least you couldn’t back then).
Various cell lysis and fluorescent dye methods were also developed at around the same time. The dye methods potentially suffer from sequestration of the dye or export from the cell. Some gave wildly different results from the NMR. However others such as by Kei Inouye (Dev Growth & Differentiation 27, 510 19985) gave similar results to NMR.
So, cytosolic pH can be measured using dyes but you have to be careful and you need to get an answer comparable to the NMR!
Rob
Robert Kay,
MRC Laboratory of Molecular Biology,
Francis Crick Avenue,
Cambridge Biomedical Campus,
Cambridge CB2 0QH,
England
Phone, Office:+ 1223 267039 (01223 267039 from UK)
Phone, Lab: + 1223 267386 (01223 267386 from UK)
FAX: + 1223 268300 (01223 268300 from UK)
Web page: https://urldefense.proofpoint.com/v2/url?u=https-3A__www2.mrc-2Dlmb.cam.ac.uk_groups_rrk&d=DwIF-g&c=yHlS04HhBraes5BQ9ueu5zKhE7rtNXt_d012z2PA6ws&r=G0i-xkKvWepiOT01FF_Nx9XkaEEFt5Dttsc3yIePxBPU44aHfHsMfVSlUygwIJiN&m=pq4XkatAXzISMFAEYp5i2uAkOiuVB6aprgiKofeg8Gk&s=hXxgHbnSndNJBFFFXEXXMnCUwcEHBNKgIkk4B1ZQCMg&e= <https://urldefense.proofpoint.com/v2/url?u=https-3A__www2.mrc-2Dlmb.cam.ac.uk_groups_rrk&d=DwMFaQ&c=yHlS04HhBraes5BQ9ueu5zKhE7rtNXt_d012z2PA6ws&r=G0i-xkKvWepiOT01FF_Nx9XkaEEFt5Dttsc3yIePxBPU44aHfHsMfVSlUygwIJiN&m=JFvWKX92NO8HLKLiv_5NX_n5CF8KA8A9iYhwJ3nTNYk&s=HdjVSLz4WaeakBeTejbBG1YmIHc2fJkVu33nSJUy4js&e=>
On 8 Oct 2019, at 15:42, Richard Gomer <[log in to unmask]<mailto:[log in to unmask]>> wrote:
Hi-
We did, many years ago, in this paper
A protein containing a serine-rich domain with vesicle fusing properties mediates cell cycle-dependent cytosolic pH regulation.
Brazill DT, Caprette DR, Myler HA, Hatton RD, Ammann RR, Lindsey DF, Brock DA, Gomer RH.
J Biol Chem. 2000 Jun 23;275(25):19231-40.
we followed cell lineages over many hours by videomicroscopy, then we gently changed to medium to medium containing the dye BCECF-AM, then imaged at high power individual cells in the field using two different fluorescence filter cubes - it was difficult....
there may be better dyes nowdays
cheers
Richard Gomer
Department of Biology
Texas A&M University
301 Old Main Drive
College Station, TX 77843-3474
979 458 5745
________________________________________
From: DICTY [[log in to unmask]<mailto:[log in to unmask]>] on behalf of Marianne Grafe [[log in to unmask]<mailto:[log in to unmask]>]
Sent: Tuesday, October 8, 2019 9:20 AM
To: [log in to unmask]<mailto:[log in to unmask]>
Subject: [DICTY] pH-sensitive dyes
Dear All,
Has anyone worked with pH-sensitive dyes to detect the cytosolic pH in
Dictyostelium?
Thanks,
Marianne
--
Marianne Grafe
PhD student
Group Cell Biology
Institute of Biochemistry and Biology
University of Potsdam
Karl-Liebknecht-Straße 24, Haus 26
14476 Potsdam
Germany
Tel.: +49-(0)331 977-5555
|
