Re: Cell permeability assay **vendor reply**

[Kilgore, Jason A.]

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**Vendor reply **

Hi, Jens,

Any reagent or compound in the cytosol that isn't bound to proteins or membranes is almost certainly going to be pumped out over time.  Often, Calcein dyes are used for membrane permeability testing, but they are lost after only about an hour or so due to active extrusion by the cell.  I don't know of a microscopy-based option that could go for days.  Gabor's idea of a fluorescent dextran might work, particularly if it is a large dextran (us as one of the 2M MW options, such as products D7137 or D7139 from Molecular Probes) which is less likely to be extruded due to size, but I'm not sure anyone has actually tested it for this sort of assay.

Another choice would be to have parallel samples at different timepoints that you assay using a non-cell-permeant nucleic acid stain, such as SYTOX Green, TO-PRO-3, or propidium iodide.

Another possibility may be a plate-based assay for G6PD release.  The cytosolic enzyme, glucose 6-phosphate dehydrogenase (G6PD), is only released by damaged cells into the surrounding medium.  You can then collect the extracellular medium at different timepoints and analyze it.  Molecular Probes has a kit to assay for G6PD, using a resazurin-to-resorufin fluorescence analysis readout.  Catalog V23111.  It's not a visual (microscopy) assay, but the data could be useful for this.

Jason

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

Thermo Fisher Scientific
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Csúcs Gábor
Sent: Thursday, August 06, 2015 2:11 PM
To: [hidden email]
Subject: Re: Cell permeability assay

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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

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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

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