http://confocal-microscopy-list.275.s1.nabble.com/Re-fixable-pH-sensor-vendor-response-and-additional-question-tp7587838p7587852.html
On Wed, Jan 24, 2018 at 1:08 PM, Kilgore, Jason A. <
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> **Vendor Response**
>
> I agree with Doug. Though the LysoSensor dextran is "fixable" in the
> sense that the dextran portion has amine groups that are crosslinked with
> formaldehyde, and will thus be retained after fixation, the pH-response is
> *not* fixable and will reflect the pH of the subsequent buffers. You could
> put the fixed cells into a pH 4 buffer to visualize the localization of the
> dextran, but not be able to see what the pH was prior to the fixation.
>
> To my knowledge, there aren't any pH indicator organic dyes that will have
> a pH *response* that will be irreversible or fixable.
>
> HOWEVER, I am not familiar with pH-sensitive fluorescent proteins. A
> question for the rest of the listserv: Would pH-responsive fluorescent
> proteins be fixable and retain the pH response?
>
> Jason
>
> Jason A. Kilgore
> Technical Application Scientist
> Molecular Probes / EVOS Tech Support
> Thermo Fisher Scientific
>
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> -----Original Message-----
> From: Confocal Microscopy List [mailto:
[hidden email]]
> On Behalf Of Douglas Richardson
> Sent: Wednesday, January 24, 2018 8:44 AM
> To:
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> Subject: Re: fixable pH sensor
>
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> Hi Sylvie,
>
> I just had this discussion with a user yesterday as well. All of the pH
> sensor that I know work based on a reversible protonation reaction that
> shifts the fluorophore's fluorescent properties. Because this reaction is
> reversible, as soon as you perforate the membrane during fixation, the pH
> probe is going to read out the pH of whatever buffer/mounting media the
> cells are sitting in.
>
> If anyone knows of an irreversible pH sensor, I'd be interested to hear
> about it!
>
> -Doug
>
> On Wed, Jan 24, 2018 at 11:04 AM, Nuno Moreno <
[hidden email]>
> wrote:
>
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> > *****
> >
> > Hi
> >
> > I see no point on measuring pH with fixed cell. Upon fixation you
> > should have an immediate dissipation of any acidic compartment,
> > specially with protons that move very fast. Even you can measure
> > something I would doubt about the quality of those results. It is hard
> > enough without fixation :)
> >
> > ...Buy a microscope for the BLS3
> >
> > regards
> > Nuno Moreno
> >
> > > On 24 Jan 2018, at 15:49, Sylvie Le Guyader
> > > <
[hidden email]>
> > wrote:
> > >
> > > *****
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https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cg> > > i-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz
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> > > include the link in your
> > posting.
> > > *****
> > >
> > > Hi Mike
> > >
> > > Yes we want to measure the Lysosome pH in cells infected with a BSL3
> > virus but we image them in a non BSL3 imaging facility so the cells
> > must be fixed before imaging.
> > >
> > > Med vänlig hälsning / Best regards
> > >
> > > Sylvie
> > >
> > > @@@@@@@@@@@@@@@@@@@@@@@@
> > > Sylvie Le Guyader, PhD
> > > Live Cell Imaging Facility Manager
> > > Karolinska Institutet- Bionut Dpt
> > > Hälsovägen 7,
> > > Novum, G lift, floor 6
> > > 14157 Huddinge
> > > Sweden
> > > mobile: +46 (0) 73 733 5008
> > > office: +46 (0) 8 524 811 72
> > > LCI website
> > >
> > >
> > > -----Original Message-----
> > > From: Confocal Microscopy List
> > > [mailto:
[hidden email]]
> > On Behalf Of MODEL, MICHAEL
> > > Sent: den 24 januari 2018 15:46
> > > To:
[hidden email]
> > > Subject: Re: fixable pH sensor
> > >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
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https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cg> > > i-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz
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https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=> > > DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQt
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> > > include the link in your
> > posting.
> > > *****
> > >
> > > Sylvie,
> > >
> > > Do you want to measure pH in chemically fixed cells? (Sorry, this is
> > > not
> > an answer to your question but a counter-question)
> > >
> > > Mike
> > >
> > > -----Original Message-----
> > > From: Confocal Microscopy List
> > > [mailto:
[hidden email]]
> > On Behalf Of Sylvie Le Guyader
> > > Sent: Wednesday, January 24, 2018 7:25 AM
> > > To:
[hidden email]
> > > Subject: fixable pH sensor
> > >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
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https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cg> > > i-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz
> > > 6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLy
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> > >
https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=> > > DwIFaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQt
> > > CbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=5V919jtGb1qMe-MAV2yv0PXy8upxs0rNnvP
> > > qFjBblhY&s=wvJ-6L87pskizMetiC6QUk2Fr08klyox7Rn-rb4fQCM&e= and
> > > include the link in your
> > posting.
> > > *****
> > >
> > > Hello everyone
> > >
> > > Would anyone know of a fixable pH sensor for lysosomes that is
> > > excited
> > in the visible range and not in the UV? We only know of the Lysosensor
> > Yellow/Blue dextran by Thermofisher we have no UV on the microscope we
> > want to use.
> > >
> > > thanks
> > >
> > > Med vänlig hälsning / Best regards
> > >
> > > Sylvie
> > >
> > > @@@@@@@@@@@@@@@@@@@@@@@@
> > > Sylvie Le Guyader, PhD
> > > Live Cell Imaging Facility Manager
> > > Karolinska Institutet- Bionut Dpt
> > > Hälsovägen 7,
> > > Novum, G lift, floor 6
> > > 14157 Huddinge
> > > Sweden
> > > mobile: +46 (0) 73 733 5008
> > > office: +46 (0) 8 524 811 72
> > > LCI
> > > website<
https://urldefense.proofpoint.com/v2/url?u=http-3A__ki.se_en> > > _bionut_welcome-2Dto-2Dthe-2Dlci-2Dfacility&d=DwIFaQ&c=q6k2DsTcEGCcC
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> >
>