Multiple Fluorescent Dyes in a Single Cell

[Adrian Bailey]

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Dear List members,

I am planning to carry out some live cell microscopy and I'm looking for some helpful tips on whether or not my dyes will be well resolved or if my experiment will work at all. A bit of background. I've synthesized an NBD labelled lipid (Green) and would like to conclusively prove that it localizes itself in the membrane. We've shown that it effects the membranes of HT-29 cells, A549 cells, and Hepa1-6 cells and any of these can be used for the microscopy experiment.

In addition to visualizing NBD, I would also like to have:
1) an F-actin dye - I plan to use SiR700 (available from Spirochrome) - Red
2) a nuclear dye - I plan to use Hoechst 33342 (Thermofischer) - Blue
3) another membrane dye - I plan to use DiR (Thermofischer) - Far Red (for co-localization)

If anyone has any insight/tips/advice I would very much appreciate it.

Cheers.

[George McNamara]

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Hi Adrian,

You could probably use Hoechst 33342 at a very low concentration to
minimize crosstalk ... for that matter, low concentrations of all the
molecules. Always useful to graph the spectra, for examples

https://www.thermofisher.com/us/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html

https://searchlight.semrock.com/

more options are listed in the Category = Fluorescence Spectra [deselect
"All", then select this category, top right from the table] at

http://www.geomcnamara.com/light-microscopy-websites

Use: to go to a site of interest, click in the far right column(s).

enjoy,
George

George McNamara, PhD
Baltimore, MD 21231
[hidden email]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75   (may need to use Microsoft Edge or Firefox, rather than Google Chrome)
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650
http://confocal.jhu.edu (as of May 22, 2017)



On 5/18/2017 12:33 PM, Adrian Bailey 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.
> *****
>
> Dear List members,
>
> I am planning to carry out some live cell microscopy and I'm looking for some helpful tips on whether or not my dyes will be well resolved or if my experiment will work at all. A bit of background. I've synthesized an NBD labelled lipid (Green) and would like to conclusively prove that it localizes itself in the membrane. We've shown that it effects the membranes of HT-29 cells, A549 cells, and Hepa1-6 cells and any of these can be used for the microscopy experiment.
>
> In addition to visualizing NBD, I would also like to have:
> 1) an F-actin dye - I plan to use SiR700 (available from Spirochrome) - Red
> 2) a nuclear dye - I plan to use Hoechst 33342 (Thermofischer) - Blue
> 3) another membrane dye - I plan to use DiR (Thermofischer) - Far Red (for co-localization)
>
> If anyone has any insight/tips/advice I would very much appreciate it.
>
> Cheers.

--

[Iain Johnson]

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*****

One thing to remember with membrane dyes like DiR, DiI etc is that membrane turnover in live mammalian cells is rapid. So if you want an image of the plasma membrane you have to do a short dye incubation and image right away. Within 30-60 mins most of the labeled membrane will be internalized.

To evaluate plasma membrane labeling, one of the best methods is destaining via back-extraction with BSA or beta-cyclodexttin. References for these techniques are in the Molecular Probes Handbook, Chapter 13.

Iain

Sent from my iPhone

> On May 20, 2017, at 9:25 AM, George McNamara <[hidden email]> 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.
> *****
>
> Hi Adrian,
>
> You could probably use Hoechst 33342 at a very low concentration to minimize crosstalk ... for that matter, low concentrations of all the molecules. Always useful to graph the spectra, for examples
>
> https://www.thermofisher.com/us/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html
>
> https://searchlight.semrock.com/
>
> more options are listed in the Category = Fluorescence Spectra [deselect "All", then select this category, top right from the table] at
>
> http://www.geomcnamara.com/light-microscopy-websites
>
> Use: to go to a site of interest, click in the far right column(s).
>
> enjoy,
> George
>
> George McNamara, PhD
> Baltimore, MD 21231
> [hidden email]
> https://www.linkedin.com/in/georgemcnamara
> https://works.bepress.com/gmcnamara/75   (may need to use Microsoft Edge or Firefox, rather than Google Chrome)
> http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650
> http://confocal.jhu.edu (as of May 22, 2017)
>
>
>
>> On 5/18/2017 12:33 PM, Adrian Bailey 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.
>> *****
>>
>> Dear List members,
>>
>> I am planning to carry out some live cell microscopy and I'm looking for some helpful tips on whether or not my dyes will be well resolved or if my experiment will work at all. A bit of background. I've synthesized an NBD labelled lipid (Green) and would like to conclusively prove that it localizes itself in the membrane. We've shown that it effects the membranes of HT-29 cells, A549 cells, and Hepa1-6 cells and any of these can be used for the microscopy experiment.
>>
>> In addition to visualizing NBD, I would also like to have:
>> 1) an F-actin dye - I plan to use SiR700 (available from Spirochrome) - Red
>> 2) a nuclear dye - I plan to use Hoechst 33342 (Thermofischer) - Blue
>> 3) another membrane dye - I plan to use DiR (Thermofischer) - Far Red (for co-localization)
>>
>> If anyone has any insight/tips/advice I would very much appreciate it.
>>
>> Cheers.
>
> --