Marianne,
We published a couple papers using the fluorescent dye pyranine in Dicty. Can follow single cells- vegetative or developing. Must get dye into cells, and stop dye from entering vesicular compartments as others noted . Our methods and findings are in two papers in JCB in 1988 and 1990. First author is Furukawa. Good luck.
Regards, marcus
> On Oct 8, 2019, at 4:36 PM, Richard Gomer <[log in to unmask]> wrote:
>
> [External Sender]
>
> 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,
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>
> 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
>
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