Posted by
George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/How-to-differentiate-plasma-membrane-vs-cytoplasm-in-Jurkat-lymphocytes-tp7586566p7586568.html
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Hi Theresa,
Jurkat are CD4 T-cells: I suggest CD4 for plasma membrane. CD3 should
also work.
I see Tim replied with fluorescence polarization (fluorescence
anisotropy). I have a different suggestion for polarization: activate
the T-cells (anti-CD3 + anti-CD28 for example ... both elongate and
'puffed up' ... optional: could coat the coverglass to get the Jurkat to
stick), and compare to the Singleton ... Wulfing Science Signaling
papers (fixed cells, antibodies to any of the proteins they used
GFP-fusions of, ought to give similar results). References below.
Consider making the Jurkat T-cells into "road kill" by squishing them
under agarose (or similar material), similar to
http://dictybase.org/techniques/pdf_docs/agar_overlay.pdfProbably too ambitious for today, but I encourage you to consider
expanding your T-cells with Expansion Microscopy,
http://expansionmicroscopy.orghttp://www.extbio.com//
singleton ... Wulfing references:
Itk controls the spatiotemporal organization of T cell activation.
Singleton KL, Gosh M, Dandekar RD, Au-Yeung BB, Ksionda O, Tybulewicz
VL, Altman A, Fowell DJ, Wülfing C.
Sci Signal. 2011 Oct 4;4(193):ra66. doi: 10.1126/scisignal.2001821.
PMID: 21971040
Spatiotemporal patterning during T cell activation is highly diverse.
Singleton KL, Roybal KT, Sun Y, Fu G, Gascoigne NR, van Oers NS, Wülfing C.
Sci Signal. 2009 Apr 7;2(65):ra15. doi: 10.1126/scisignal.2000199.
PMID: 19351954
A large T cell invagination with CD2 enrichment resets receptor
engagement in the immunological synapse.
Singleton K, Parvaze N, Dama KR, Chen KS, Jennings P, Purtic B, Sjaastad
MD, Gilpin C, Davis MM, Wülfing C.
J Immunol. 2006 Oct 1;177(7):4402-13. PMID: 16982875
George
On 3/6/2017 1:47 PM, Swayne, Theresa C. wrote:
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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.
>
>
> ------------------------------------
> 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: 212-851-4613
>
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George McNamara, PhD
Houston, TX 77054
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https://www.linkedin.com/in/georgemcnamarahttps://works.bepress.com/gmcnamara/75/http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650