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

Posted by Renato A. Mortara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/How-to-differentiate-plasma-membrane-vs-cytoplasm-in-Jurkat-lymphocytes-tp7586566p7586569.html

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My suggestion is to label live cells (in ice) with the anti-epitope.

Renato

-----Mensagem original-----
De: Confocal Microscopy List [mailto:[hidden email]] Em nome de Swayne, Theresa C.
Enviada em: segunda-feira, 6 de março de 2017 16:48
Para: [hidden email]
Assunto: 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
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