How to show something inside the cell?

[SUBSCRIBE CONFOCALMICROSCOPY Sara Smith]

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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
 

[mmodel]

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
 

[Gabriel Lapointe-4]

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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
7141 Sherbrooke St. West SP 534
Montréal QC H4B 1R6 Canada
Lab : [phone] x5988
Office : [phone] x3008
Fax : [phone]
Cell : [phone]
[hidden email]
cmac.concordia.ca
http://gabriellapointe.ca


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?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
>
>
>

[Stephen Firth (Med)]

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Another possibility is using a membrane lawn, sorry can't remember the
reference. Essentially run the experiment, staining etc but shear the cell
off the coverslip leaving the membrane behind. If the target is on the
membrane it will be visible, but if it has moved through into the cytoplasm
or beyond it will have been removed.
Cheers,
Stephen


On 7 June 2013 07:17, Gabriel Lapointe <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
> 7141 Sherbrooke St. West SP 534
> Montréal QC H4B 1R6 Canada
> Lab : [phone] x5988
> Office : [phone] x3008
> Fax : [phone]
> Cell : [phone]
> [hidden email]
> cmac.concordia.ca
> http://gabriellapointe.ca
>
>
> 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?
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > 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
> >
> >
> >
>

--
Stephen Firth
Manager of Advanced Optical Microscopy
Monash Micro Imaging
Monash University
Room G53A Building 75
Phone [phone]
Mob [phone]

[mmodel]

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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
7141 Sherbrooke St. West SP 534
Montréal QC H4B 1R6 Canada
Lab : [phone] x5988
Office : [phone] x3008
Fax : [phone]
Cell : [phone]
[hidden email]
cmac.concordia.ca
http://gabriellapointe.ca


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?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
>
>
>

[lechristophe]

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On a related note:

Bromophenol blue (BPB) was used at 0.5 - 2 mM to quench extracellular GFP
fluorescence from fusion proteins (see Harata... Tsien Neuron 2006 and
Song... Poo Cell 2009). However in our hands (we tried only once), it lead
to a dramatic photosensibilization of cultured neurons, they would just die
very rapidly under reasonable imaging conditions, and we weren't able to
use this method. I'd be happy to hear about people that successively used
BPB for this purpose.

--
Christophe Leterrier
Researcher
Axonal Domains Architecture Team
CRN2M CNRS UMR 7286
Aix Marseille University, France



On Fri, Jun 7, 2013 at 1:03 PM, MODEL, MICHAEL <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
> 7141 Sherbrooke St. West SP 534
> Montréal QC H4B 1R6 Canada
> Lab : [phone] x5988
> Office : [phone] x3008
> Fax : [phone]
> Cell : [phone]
> [hidden email]
> cmac.concordia.ca
> http://gabriellapointe.ca
>
>
> 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?
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > 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
> >
> >
> >
>
>

[Kavita Aswani-2]

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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. [phone]| Cell. 647 290 3506 * Toll Free (USA and Canada) 1 800 668-8752
[hidden email] * http://www.ldgi.com/

-----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
7141 Sherbrooke St. West SP 534
Montréal QC H4B 1R6 Canada
Lab : [phone] x5988
Office : [phone] x3008
Fax : [phone]
Cell : [phone]
[hidden email]
cmac.concordia.ca
http://gabriellapointe.ca


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?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
>
>
>

Think Green Before You Print!

This email and any attachments thereto may contain private, confidential, and privileged material for the sole use of the intended recipient. Any review, copying, or distribution of this email (or any attachments thereto) by others is strictly prohibited. If you are not the intended recipient, please contact the sender immediately, and permanently delete the original and any copies of this email and any attachments thereto.

[Kilgore, Jason-2]

**vendor reply**

A common method would be to use a quencher in the cell media, to quench any surface fluorescence.

A traditional method would be to use trypan blue.  However, this is very broad-range -- a bad thing if you wish to quench only a selected dye in a multiplex situation

Perhaps a better method would be to use one of the BackDrop fluorescence quenching reagents, which can be targeted to blue, green or red.  And they come in handy dropper-bottle formulations.  Link: https://products.invitrogen.com/ivgn/product/R37603?ICID=search-product

Any dye that is internalized in live cells, with either of these options, will be fluorescent.

Cheers,

Jason

Jason A. Kilgore
Technical Application Scientist
Molecular Probes Labeling and Detection Technologies
Cells Systems Division
 
T 1 800 955 6288 then option 4, then option 6,  or  541 335 0353 • F 541 335 0238
29851 Willow Creek Rd • Eugene • OR • 97402-9132 • United States



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of SUBSCRIBE CONFOCALMICROSCOPY
Sent: Thursday, June 06, 2013 1:27 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
 

[Mike Ignatius-2]

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Hi Yi,

In a further evolution of the quencher approach consider this paper:

Angew Chem Int Ed Engl. 2013 May 27;52(22):5744-8. doi: 10.1002/anie.201301243. Epub 2013 Apr 19.
A programmable sensor to probe the internalization of proteins and nanoparticles in live cells.
Liu H, Johnston AP.

Instead of bathing the whole prep in quencher, with its associated off target concerns, a complimentary nucleic acid probe is made with a quencher.  Much more specific and pretty easy to make.

Mike Ignatius
Marker Gene Tech

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of MODEL, MICHAEL
Sent: Thursday, June 06, 2013 1:34 PM
To: [hidden email]
Subject: Re: How to show something inside the cell?

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?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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
 

[SUBSCRIBE CONFOCALMICROSCOPY Sara Smith]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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. [phone]| Cell. 647 290 3506 * Toll Free (USA and Canada) 1
> 800 668-8752
> [hidden email] * http://www.ldgi.com/
>
> -----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?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
> 7141 Sherbrooke St. West SP 534
> Montréal QC H4B 1R6 Canada
> Lab : [phone] x5988
> Office : [phone] x3008
> Fax : [phone]
> Cell : [phone]
> [hidden email]
> cmac.concordia.ca
> http://gabriellapointe.ca
>
>
> 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?
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > 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
> >
> >
> >
>
> Think Green Before You Print!
>
> This email and any attachments thereto may contain private, confidential,
> and privileged material for the sole use of the intended recipient. Any
> review, copying, or distribution of this email (or any attachments thereto)
> by others is strictly prohibited. If you are not the intended recipient,
> please contact the sender immediately, and permanently delete the original
> and any copies of this email and any attachments thereto.
>

[mmodel]

*****
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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:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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.
> 905 812-3342| Cell. 647 290 3506 * Toll Free (USA and Canada) 1
> 800 668-8752
> [hidden email] * http://www.ldgi.com/
>
> -----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?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
> 7141 Sherbrooke St. West SP 534
> Montréal QC H4B 1R6 Canada
> Lab : [phone] x5988
> Office : [phone] x3008
> Fax : [phone]
> Cell : [phone]
> [hidden email]
> cmac.concordia.ca
> http://gabriellapointe.ca
>
>
> 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?
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > 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
> >
> >
> >
>
> Think Green Before You Print!
>
> This email and any attachments thereto may contain private,
> confidential, and privileged material for the sole use of the intended
> recipient. Any review, copying, or distribution of this email (or any
> attachments thereto) by others is strictly prohibited. If you are not
> the intended recipient, please contact the sender immediately, and
> permanently delete the original and any copies of this email and any attachments thereto.
>

[Kilgore, Jason-2]

*****
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Eventually, the quenchers in the media will be internalized in live cells, simply due to repeated endocytotic uptake.  Usually you have at least 45-60 minutes before this is a problem, but it depends on the rate of endocytosis of your cell line.

If you cells die or in some other way have the membranes compromised, then certainly the external quencher will now quench internally as well.  As one other commenter pointed out, this isn't an issue if you are only wanting to image live cells, though.

Cheers,

Jason


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Yi Zheng
Sent: Friday, June 07, 2013 1:07 PM
To: [hidden email]
Subject: Re: How to show something inside the cell?

*****
To join, leave or search the confocal microscopy listserv, go to:
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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:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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. [phone]| Cell. 647 290 3506 * Toll Free (USA and Canada) 1
> 800 668-8752
> [hidden email] * http://www.ldgi.com/
>
> -----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?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
> 7141 Sherbrooke St. West SP 534
> Montréal QC H4B 1R6 Canada
> Lab : [phone] x5988
> Office : [phone] x3008
> Fax : [phone]
> Cell : [phone]
> [hidden email]
> cmac.concordia.ca
> http://gabriellapointe.ca
>
>
> 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?
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > 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
> >
> >
> >
>
> Think Green Before You Print!
>
> This email and any attachments thereto may contain private, confidential,
> and privileged material for the sole use of the intended recipient. Any
> review, copying, or distribution of this email (or any attachments thereto)
> by others is strictly prohibited. If you are not the intended recipient,
> please contact the sender immediately, and permanently delete the original
> and any copies of this email and any attachments thereto.
>

[Russell M. Taylor II]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Once you have the 3D data, you can use ImageSurfer (download from
www.cismm.org) to load and isosurface it, or volume-render it, or
slice through it along arbitrary axes so that you can see the 3D
layout.  This will help you confirm the distribution is inside the
cell without having to look through slice by slice and mentally
reconstruct the 3D distributions.


---
Russell M. Taylor II, Ph.D.                           [hidden email]
CB #3175, Sitterson Hall                        www.cs.unc.edu/~taylorr
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