Re: How to show something inside the cell?

Posted by mmodel on
URL: http://confocal-microscopy-list.275.s1.nabble.com/How-to-show-something-inside-the-cell-tp7580451p7580464.html

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But you are probably not interested in looking at already dead cells. And quenchers act very fast. Moderate lowering of pH, trypan blue or acid blue are not going to harm the cells.


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Yi Zheng
Sent: Friday, June 07, 2013 4:07 PM
To: [hidden email]
Subject: Re: How to show something inside the cell?

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Thank you for all valuable inputs. We can certainly try to quench the fluorophore on the cell surface. However, I am wondering if the quencher can penetrate the cell membrane and get into the cells if cells are dying or incubated with the quenchers for too long. Some treatment seem quite harsh to me.





On Fri, Jun 7, 2013 at 9:35 AM, Kavita Aswani <[hidden email]>wrote:

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> I can confirm Michaels comment below.  During my research life, I was
> labelling bacteria with FITC, and observing uptake by cultured macrophages.
>  Due to the conditions used, a lot of these FITC-bugs stuck to the
> outside of the cell, and I used Typan blue to quench the extracellular
> fluorescence.  Details in http://www.ncbi.nlm.nih.gov/pubmed/11726395
>
> TITLE: Effects of surfactant protein A and NaCl concentration on the
> uptake of Pseudomonas aeruginosa by THP-1 cells. Khubchandani-Aswani
> KR, Oberley RE, Snyder JM.
>
> Cheers,
> Kavita
>
> Kavita Aswani, PhD
> Senior Applications Scientist - Life Sciences Lumen Dynamics Group
> Inc.
> 2260 Argentia Road, Mississauga, Ontario, Canada, L5N 6H7 Direct Tel.
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> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]]
> On Behalf Of MODEL, MICHAEL
> Sent: June-07-13 7:04 AM
> To: [hidden email]
> Subject: Re: How to show something inside the cell?
>
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> I think trypan blue has also been used to quench external fluorescence
> ________________________________________
> From: Confocal Microscopy List [[hidden email]] on
> behalf of Gabriel Lapointe [[hidden email]]
> Sent: Thursday, June 06, 2013 5:17 PM
> To: [hidden email]
> Subject: Re: How to show something inside the cell?
>
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> Hi Yi,
> In the same line of thougth as Mike, you could also look at the
> quencher used in qPCR. Some might be compatible with your fluorophore.
>
>
>
> *Gabriel Lapointe, M.Sc.*
> Lab Manager / Microscopy Specialist
> Concordia University, Biology Department
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> 2013/6/6 MODEL, MICHAEL <[hidden email]>
>
> > Hi Yi - I think your solution is good. Fluorophores on the surface
> > can also be selectively quenched, depending on the fluorophore -
> > FITC by lowering pH, red fluorophores or those emitting in the 600s
> > by adding several mg/ml of Acid Blue 9.
> >
> > Mike Model
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List
> > [mailto:[hidden email]]
> > On Behalf Of SUBSCRIBE CONFOCALMICROSCOPY
> > Sent: Thursday, June 06, 2013 4:27 PM
> > To: [hidden email]
> > Subject: How to show something inside the cell?
> >
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> > Dear listers,
> >
> > One of our user is investigating the transportation of fluorescence
> > labeled DNA oligo in cells. He would like to prove the DNA oligo has
> > been transported inside the cells but not on the surface of cells by
> > using the light microscopy. We suggested him to clearly label the
> > membrane of cells and use the Z-stack images of confocal to look
> > through
> the cells.
> > I am wondering if there is any other better solution?
> >
> > Any suggestion on good membrane dye is appreciated as well!
> >
> > Thank you,
> >
> > Yi
> >
> >
> >
>
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