Re: fluorescein diacetate (fda) ** vendor response **

[Kilgore, Jason A.]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****


** vendor response **

Yes, any amount of water will hydrolyze the diacetate. The stock probably got hydrated.

This can include the tiny amount absorbed in DMSO, which is more typically used to make the stock rather than acetone (even small amounts of acetone after dilution can be injurious to cells, but cells are more resistant to DMSO).

Here at Molecular Probes Tech Support, we actually recommend performing a dry down aliquoting for long term storage rather than hoping the DMSO or other solvent is truly anhydrous.

For FDA and CMFDA, we recommend bringing up the stock in CHCl3:MeOH mix, CHCl3, MeCN or Ethylacetate as the solvent, at 1 mg/mL, then aliquot into single-use tubes (such as PCR tubes), then put the tubes into a vacuum bell or chamber and pull a vacuum on it with house vacuum or a vacuum pump (and tubing that is compatible with these solvents). Once the solvent has evaporated off and it is back to powder, cap and seal the tubes, put them in a baggie with a desiccant pack, and store at -20 until ready to use.

Jason

Jason A. Kilgore
Technical Application Scientist
Molecular Probes / EVOS Tech Support
Life Sciences Solutions
 

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Jacqueline Ross
Sent: Tuesday, April 20, 2021 2:58 PM
To: [hidden email]
Subject: Re: fluorescein diacetate (fda)

CAUTION: This email originated from outside of Thermo Fisher Scientific. If you believe it to be suspicious, report using the Report Phish button in Outlook or send to [hidden email].


*****
To join, leave or search the confocal microscopy listserv, go to:
https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIGaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=x4ff_7MOZr3rQ1PN5F9h5PEyeScLGCyXtN8D1ARslZY&s=yRhGFh2MZFEpaEfE7ouG9aIn8cuU4tsx4kYNOrgmKco&e=
Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIGaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=x4ff_7MOZr3rQ1PN5F9h5PEyeScLGCyXtN8D1ARslZY&s=rVkD8dT6JzEVoE9bNdoxrUSYaaZgpi7vRspEPk8lGbE&e=  and include the link in your posting.
*****

Hi Aryeh,

For CMDFA, we make up the stock solution in dry DMSO as per the instruction sheet and then store in aliquots at -20 degrees Celsius. If it gets any water in it, it won’t work as it hydrolyses so we make sure to use freezer tubes that seal really tight, rather than standard Eppendorfs. It might be the same for FDA.

Cheers,

Jacqui

From: Confocal Microscopy List <[hidden email]> On Behalf Of Aryeh Weiss
Sent: Tuesday, April 20, 2021 9:33 PM
To: [hidden email]
Subject: fluorescein diacetate (fda)

*****
To join, leave or search the confocal microscopy listserv, go to:
https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIGaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=x4ff_7MOZr3rQ1PN5F9h5PEyeScLGCyXtN8D1ARslZY&s=yRhGFh2MZFEpaEfE7ouG9aIn8cuU4tsx4kYNOrgmKco&e= <https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIGaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=x4ff_7MOZr3rQ1PN5F9h5PEyeScLGCyXtN8D1ARslZY&s=yRhGFh2MZFEpaEfE7ouG9aIn8cuU4tsx4kYNOrgmKco&e= > Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIGaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=x4ff_7MOZr3rQ1PN5F9h5PEyeScLGCyXtN8D1ARslZY&s=rVkD8dT6JzEVoE9bNdoxrUSYaaZgpi7vRspEPk8lGbE&e= <https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIGaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=x4ff_7MOZr3rQ1PN5F9h5PEyeScLGCyXtN8D1ARslZY&s=rVkD8dT6JzEVoE9bNdoxrUSYaaZgpi7vRspEPk8lGbE&e= > and include the link in your posting.
*****

I am using fluorescein diacetate in my teaching lab. The student get to see the cells light up, measure the increase over time (takes a minute or two), see it photobleach afterwards, and  process it by subtracting the in-cell signal from background fluorescence. Very nice.

My question concerns the storage of the fda. We made a 0.5mM stock solution in acetone, which worked great with a further 1/200 dilution into the plate. We could probably have also diluted 1/1000.
We stored the leftover stock solution at -20C. A week later that stock solution did not work at all. When we added 10ul to cell culture plate , we saw an increase in background, but absolutely nothing in the cells.

We then made a fresh stock solution of fda, and it worked perfectly.
Exactly as expected.

So my question is - what happened?

Is there  a trick to keeping the stock solution active, or do we need to make it fresh every week?

Thanks for any advise you can provide.

--aryeh

--
Aryeh Weiss
Faculty of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph: 972-3-5317638
FAX: [phone]

[Aryeh Weiss]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Thank you to all those who replied, both on-list and off-list.

The consensus is that we need to make sure there is no water in the
solvent.

However, if the fda was hydrolyzed, I would have expected that tube of
stock solution to be highly fluorescent, and it was not.
One response suggested that the dye was in the leuco form.

Are there derivatives of fda that are more stable in the presence of
small amounts of water?

Best regards
--aryeh



On 21/04/2021 01:30, Kilgore, Jason A. wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
>
> ** vendor response **
>
> Yes, any amount of water will hydrolyze the diacetate. The stock probably got hydrated.
>
> This can include the tiny amount absorbed in DMSO, which is more typically used to make the stock rather than acetone (even small amounts of acetone after dilution can be injurious to cells, but cells are more resistant to DMSO).
>
> Here at Molecular Probes Tech Support, we actually recommend performing a dry down aliquoting for long term storage rather than hoping the DMSO or other solvent is truly anhydrous.
>
> For FDA and CMFDA, we recommend bringing up the stock in CHCl3:MeOH mix, CHCl3, MeCN or Ethylacetate as the solvent, at 1 mg/mL, then aliquot into single-use tubes (such as PCR tubes), then put the tubes into a vacuum bell or chamber and pull a vacuum on it with house vacuum or a vacuum pump (and tubing that is compatible with these solvents). Once the solvent has evaporated off and it is back to powder, cap and seal the tubes, put them in a baggie with a desiccant pack, and store at -20 until ready to use.
>
> Jason
>
> Jason A. Kilgore
> Technical Application Scientist
> Molecular Probes / EVOS Tech Support
> Life Sciences Solutions
>  
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On Behalf Of Jacqueline Ross
> Sent: Tuesday, April 20, 2021 2:58 PM
> To: [hidden email]
> Subject: Re: fluorescein diacetate (fda)
>
> CAUTION: This email originated from outside of Thermo Fisher Scientific. If you believe it to be suspicious, report using the Report Phish button in Outlook or send to [hidden email].
>
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIGaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=x4ff_7MOZr3rQ1PN5F9h5PEyeScLGCyXtN8D1ARslZY&s=yRhGFh2MZFEpaEfE7ouG9aIn8cuU4tsx4kYNOrgmKco&e=
> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIGaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=x4ff_7MOZr3rQ1PN5F9h5PEyeScLGCyXtN8D1ARslZY&s=rVkD8dT6JzEVoE9bNdoxrUSYaaZgpi7vRspEPk8lGbE&e=  and include the link in your posting.
> *****
>
> Hi Aryeh,
>
> For CMDFA, we make up the stock solution in dry DMSO as per the instruction sheet and then store in aliquots at -20 degrees Celsius. If it gets any water in it, it won’t work as it hydrolyses so we make sure to use freezer tubes that seal really tight, rather than standard Eppendorfs. It might be the same for FDA.
>
> Cheers,
>
> Jacqui
>
> From: Confocal Microscopy List <[hidden email]> On Behalf Of Aryeh Weiss
> Sent: Tuesday, April 20, 2021 9:33 PM
> To: [hidden email]
> Subject: fluorescein diacetate (fda)
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIGaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=x4ff_7MOZr3rQ1PN5F9h5PEyeScLGCyXtN8D1ARslZY&s=yRhGFh2MZFEpaEfE7ouG9aIn8cuU4tsx4kYNOrgmKco&e= <https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIGaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=x4ff_7MOZr3rQ1PN5F9h5PEyeScLGCyXtN8D1ARslZY&s=yRhGFh2MZFEpaEfE7ouG9aIn8cuU4tsx4kYNOrgmKco&e= > Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIGaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=x4ff_7MOZr3rQ1PN5F9h5PEyeScLGCyXtN8D1ARslZY&s=rVkD8dT6JzEVoE9bNdoxrUSYaaZgpi7vRspEPk8lGbE&e= <https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIGaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=x4ff_7MOZr3rQ1PN5F9h5PEyeScLGCyXtN8D1ARslZY&s=rVkD8dT6JzEVoE9bNdoxrUSYaaZgpi7vRspEPk8lGbE&e= > and include the link in your posting.
> *****
>
> I am using fluorescein diacetate in my teaching lab. The student get to see the cells light up, measure the increase over time (takes a minute or two), see it photobleach afterwards, and  process it by subtracting the in-cell signal from background fluorescence. Very nice.
>
> My question concerns the storage of the fda. We made a 0.5mM stock solution in acetone, which worked great with a further 1/200 dilution into the plate. We could probably have also diluted 1/1000.
> We stored the leftover stock solution at -20C. A week later that stock solution did not work at all. When we added 10ul to cell culture plate , we saw an increase in background, but absolutely nothing in the cells.
>
> We then made a fresh stock solution of fda, and it worked perfectly.
> Exactly as expected.
>
> So my question is - what happened?
>
> Is there  a trick to keeping the stock solution active, or do we need to make it fresh every week?
>
> Thanks for any advise you can provide.
>
> --aryeh
>
> --
> Aryeh Weiss
> Faculty of Engineering
> Bar Ilan University
> Ramat Gan 52900 Israel
>
> Ph: 972-3-5317638
> FAX: [phone]

--
Aryeh Weiss
Faculty of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph:  972-3-5317638
FAX: [phone]