Cell permeability assay

[Jens]

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Dear List,

I would like to image the spread of a virus through a monolayer. The virus is fluorescently tagged such that infected cells can be easily followed.
At some point late in infection, cells will loose their membrane integrity and I would like to record that timepoint.
Now comes my question: Does anybody know a cell permeability marker that can be left in the culture medium for days and does not affect the cells? Trypan Blue?

Thanks!

Jens

--------------------------------------------------------------
Jens B. Bosse Ph.D.
Enquist Lab
Department of Molecular Biology
and
Princeton Neuroscience Institute
Princeton University
301 Schultz Lab
Washington Rd
08544 Princeton, NJ, USA

Phone: [phone]
Email: [hidden email]
Web: http://molbio.princeton.edu/labs/enquist/

This electronic communication, including any attached documents, may contain confidential and/or legally privileged information that is intended only for use by the recipient(s) named above.  If you have received this communication in error, please notify the sender immediately and delete the communication and any attachments.

[Csúcs Gábor-3]

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Dextran? - Just an idea...

Greetings   Gabor

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jens-Bernhard Bosse
Sent: Thursday, August 6, 2015 10:42 PM
To: [hidden email]
Subject: Cell permeability assay

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Dear List,

I would like to image the spread of a virus through a monolayer. The virus is fluorescently tagged such that infected cells can be easily followed.
At some point late in infection, cells will loose their membrane integrity and I would like to record that timepoint.
Now comes my question: Does anybody know a cell permeability marker that can be left in the culture medium for days and does not affect the cells? Trypan Blue?

Thanks!

Jens

--------------------------------------------------------------
Jens B. Bosse Ph.D.
Enquist Lab
Department of Molecular Biology
and
Princeton Neuroscience Institute
Princeton University
301 Schultz Lab
Washington Rd
08544 Princeton, NJ, USA

Phone: [phone]
Email: [hidden email]
Web: http://molbio.princeton.edu/labs/enquist/

This electronic communication, including any attached documents, may contain confidential and/or legally privileged information that is intended only for use by the recipient(s) named above.  If you have received this communication in error, please notify the sender immediately and delete the communication and any attachments.

[mmodel]

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Acid blue 9 (TCI America) can be left for 24 hours, maybe longer. it doesn't affect anything at up to 5-10 mg/ml and doesn't get significantly endocytosed either. We haven't tested it for much longer times but I guess it should be safe if you reduce its concentration. We use it mostly in transmission setup but it is also fluorescent at a ~630 nm excitation. If the virus is green fluorescent it will not be quenched.

Mike Model

________________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Jens-Bernhard Bosse <[hidden email]>
Sent: Thursday, August 6, 2015 4:42 PM
To: [hidden email]
Subject: Cell permeability assay

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

Dear List,

I would like to image the spread of a virus through a monolayer. The virus is fluorescently tagged such that infected cells can be easily followed.
At some point late in infection, cells will loose their membrane integrity and I would like to record that timepoint.
Now comes my question: Does anybody know a cell permeability marker that can be left in the culture medium for days and does not affect the cells? Trypan Blue?

Thanks!

Jens

--------------------------------------------------------------
Jens B. Bosse Ph.D.
Enquist Lab
Department of Molecular Biology
and
Princeton Neuroscience Institute
Princeton University
301 Schultz Lab
Washington Rd
08544 Princeton, NJ, USA

Phone:  [phone]
Email:  [hidden email]
Web:    http://molbio.princeton.edu/labs/enquist/

This electronic communication, including any attached documents, may contain confidential and/or legally privileged information that is intended only for use by the recipient(s) named above.  If you have received this communication in error, please notify the sender immediately and delete the communication and any attachments.

[Knecht, David]

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I would suggest a non-permeant DNA binding dye like PI, EtBr or Hoechst (depending on whether your cells exclude it).  Some of the Life Tech Syto dyes are also non-permeant depending on cell type.  DNA is so concentrated in the nucleus that a low concentration in the medium will be highly concentrated in the nucleus and light up permeable cells.  Dave

On Aug 6, 2015, at 4:42 PM, Jens-Bernhard Bosse <[hidden email]<mailto:[hidden email]>> wrote:

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Post images on http://www.imgur.com and include the link in your posting.
*****

Dear List,

I would like to image the spread of a virus through a monolayer. The virus is fluorescently tagged such that infected cells can be easily followed.
At some point late in infection, cells will loose their membrane integrity and I would like to record that timepoint.
Now comes my question: Does anybody know a cell permeability marker that can be left in the culture medium for days and does not affect the cells? Trypan Blue?

Thanks!

Jens

--------------------------------------------------------------
Jens B. Bosse Ph.D.
Enquist Lab
Department of Molecular Biology
and
Princeton Neuroscience Institute
Princeton University
301 Schultz Lab
Washington Rd
08544 Princeton, NJ, USA

Phone: [phone]
Email: [hidden email]
Web: http://molbio.princeton.edu/labs/enquist/

This electronic communication, including any attached documents, may contain confidential and/or legally privileged information that is intended only for use by the recipient(s) named above.  If you have received this communication in error, please notify the sender immediately and delete the communication and any attachments.

Dr. David Knecht
Professor of Molecular and Cell Biology
Core Microscopy Facility Director
University of Connecticut
91 N. Eagleville Rd.
Storrs, CT 06269
860-486-2200

[Arvydas Matiukas]

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Hello list,
 
There were posts on successfully restoring computer  and software
for  DeltaVision DV/U series microscope without dealing with GE Healthcare.
 
 We got a hardware problem: on our DeltaVision Core and personal DV
system  excitation filter wheel motor's encoder  does not  initialize
correctly, and recently MIC module no longer powers up.
The sad part of this story that in 2014 inexperienced engineer was sent by GE
and during system move damaged some hardware, and was not able to
restore the system to working condition.
 
Please share any useful resources to help us resolve our problems.
The first preference would be to find a third party service company,
though any relevant documention, or advice on self-servicing and
getting/fixing mentioned parts is appreciated as well.
It is OK to reply off the list.
 
Thanks.
 
Arvydas
====================
 
 
 
 
Arvydas Matiukas, Ph.D.
Director of Confocal&Two-Photon Core
SUNY Upstate Medical University
Neuroscience & Physiology Dept

Room 4607 IHP
505 Irving Ave
Syracuse, NY 13210
Email: [hidden email]

[Mel Symeonides]

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Hello Arvydas,

I wrote up that software restoration post for the DV/U (anyone, please
feel free to contact me if you are attempting it and would like help).
Our DV has been functioning perfectly since I did that (better than
ever, really... CentOS 6 is great!).

I think your best option is to go back to GE and open a support ticket,
and keep asking to be escalated until you get in direct contact with
someone at the Seattle office (i.e. where the "original" API offices are
located). It took a few weeks, but we were eventually put in touch with
someone there who was very understanding, extremely helpful and
knowledgeable, and ultimately resolved our hardware issue (that,
similarly to your case, was partially caused by a supposedly experienced
GE engineer who has since left GE) at zero cost to us.

This was not the workstation computer HDD crash issue (they could not
offer us any help for that other than some tips on getting the software
to work on a fresh installation, or the option to purchase a new
computer for a ridiculous price) but a separate issue that had to do
with our Xenon light source causing shutdowns for the IC/MIC. They
initially thought it might be a grounding issue, so they sent us a
(free) grounding kit with instructions, but it did not solve the
problem. They ended up sending us a separate power supply unit just for
the light source, a part that I'm pretty sure would cost many thousands
of dollars if purchased normally, and this worked perfectly. Given that
we had already paid that much off-contract (a couple of years earlier)
to have this very issue serviced, and the engineer left claiming it had
been resolved when it in fact hadn't, I think they handled it as well as
anyone could by giving us this part for free.

I would really suggest that you try that before you go to a third party
engineer. If you can clearly explain the history of the hardware fault
and provide any documentation or records you might have, I think you
should be able to have this resolved at low or no cost to you, as long
as you can get in touch with Seattle. I obviously can't guarantee that
you will receive free service or free parts, but it is well worth the try.

I'm not sure about providing the name of the person in Seattle who
helped us, but if you've tried your best and gotten nowhere, I will
provide it to you privately.

Best of luck!
Mel


On 8/10/2015 4:56 PM, Arvydas Matiukas 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.
> *****
>
> Hello list,
>
> There were posts on successfully restoring computer  and software
> for  DeltaVision DV/U series microscope without dealing with GE Healthcare.
>
>   We got a hardware problem: on our DeltaVision Core and personal DV
> system  excitation filter wheel motor's encoder  does not  initialize
> correctly, and recently MIC module no longer powers up.
> The sad part of this story that in 2014 inexperienced engineer was sent by GE
> and during system move damaged some hardware, and was not able to
> restore the system to working condition.
>
> Please share any useful resources to help us resolve our problems.
> The first preference would be to find a third party service company,
> though any relevant documention, or advice on self-servicing and
> getting/fixing mentioned parts is appreciated as well.
> It is OK to reply off the list.
>
> Thanks.
>
> Arvydas
> ====================
>
>
>
>
> Arvydas Matiukas, Ph.D.
> Director of Confocal&Two-Photon Core
> SUNY Upstate Medical University
> Neuroscience & Physiology Dept
>
> Room 4607 IHP
> 505 Irving Ave
> Syracuse, NY 13210
> Email: [hidden email]
>

--
Menelaos Symeonides
University of Vermont
Cell & Molecular Biology Graduate Program
Department of Microbiology and Molecular Genetics
318 Stafford Hall
95 Carrigan Dr
Burlington, VT 05405
[hidden email]
Phone: [phone]