Re: How to differentiate plasma membrane vs. cytoplasm in Jurkat lymphocytes?

Posted by darren.thomson on
URL: http://confocal-microscopy-list.275.s1.nabble.com/How-to-differentiate-plasma-membrane-vs-cytoplasm-in-Jurkat-lymphocytes-tp7586566p7586584.html

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Hi Theresa,

CellMask gets internalised in living cells (~ 40mins to start endocytosing, works nicely up to this point). In macrophages we've seen that fixing them seems to cause the CellMask to internalise, whether we add Cellmask before or after fixation. But this still curiously allows us to surface render the Macs quite nicely in IMARIS.

I'd be very interested to hear of a live-cell PM marker that doesn't internalise...

Dr Darren Thomson | Experimental Officer
Manchester Fungal Infection Group (MFIG)
Tel: (+44) (0)161 306 2457
Email: [hidden email]
 

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Swayne, Theresa C.
Sent: 06 March 2017 19:48
To: [hidden email]
Subject: How to differentiate plasma membrane vs. cytoplasm in Jurkat lymphocytes?

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Dear fellow confocalists,

One of my users would like to determine whether an epitope-tagged protein, expressed in Jurkat cells, is localized to the plasma membrane or the cytosol. These cells have only a thin layer of cytoplasm between the nucleus and PM.

It seems to me that at minimum we need a good plasma membrane marker, and control proteins known to be in cytosol and plasma membrane respectively.  Then I’m thinking we could do scanning confocal with high NA lens, and profile analysis or overlap analysis to measure the degree of coincidence between the membrane marker and the tagged protein.   Does that make sense as a strategy?

If so, do folks have a preferred membrane marker for this cell type? I’ve seen things like CellMask and PKH26, but the images I’ve seen online and in other cell types don’t show “pure” plasma membrane labeling.

Thanks in advance for any suggestions.


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Theresa Swayne, Ph.D.
Manager
Confocal and Specialized Microscopy Shared Resource<http://hiccc.columbia.edu/research/sharedresources/confocal>

Herbert Irving Comprehensive Cancer Center Columbia University Medical Center
1130 St. Nicholas Ave., Room 222A
New York, NY 10032
Phone: [phone]
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