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

Posted by Sylvie Le Guyader on
URL: http://confocal-microscopy-list.275.s1.nabble.com/How-to-differentiate-plasma-membrane-vs-cytoplasm-in-Jurkat-lymphocytes-tp7586566p7586582.html

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Hi Theresa

'localized to the plasma membrane': It could be inserted in, attached to or accumulated near the membrane. Differentiating between these states requires other techniques. You may get away with SIM.

If the question is instead: is there more protein in the region of the cytoplasm that is closer to the membrane -let's say half the cytoplasm thickness- compared to the portion near the nucleus, then you need to fit more than 4 pixels in the cytoplasm to be able to separate the 2 cytoplasmic halves.

With a confocal, short wavelength and 1.4NA objective you can manage if the cytoplasm is thicker than 400ish nm. If the cell is 6um in radius and the nucleus takes 95% of the volume, you are left with a 300nm thick cytoplasm... So success depends on the cytoplasm thickness.

Considering the equipment you already have, I would start as you suggest with labelling the cytoplasm in green. I would not stain the membrane to start with.

I would use a bright blue antibody like the Brilliant Violet dyes, to stain your protein of interest, and as you mentioned, known cytoplasmic and several membrane (inserted, attached, near) controls labelled in the same way.

Sample preparation and objective choice becomes crucial: coverslip #1.5h, especially if your objective has a correction ring (which must be set as well), sample attached to the coverslip, matching RIs.

See if your cells attach to poly-lysin coated coverslips. Cleaning the coverslip thoroughly with fuming HCl and NaOH before coating helps. If not, stain in a tube then cytospin on a coverslip attached to a glass slide so that the cells end up on the coverslip. Then mount the coverslip on a slide matching objective and mounting medium RIs.

If you have a hardware autofocus and the cells are almost all the same size which is often the case for blood cells, find out which offset gets you the focus at the equator. Image with no saturation or underexposure at all a thin equatorial section at Nyquist sampling with a pinhole 0.7-1 AU for the blue if possible to get as low out of focus contribution. Analyse the images automatically. Without hardware autofocus, do a small Nyquist z stack around the equator and let the software decide where the equator is.

Segment the cytoplasm. If the protein of interest is concentrated near the membrane you should get a lower blue intensity in the half of cytoplasm closer to the membrane compared to the half near the nucleus.

Voilá :-)

Med vänlig hälsning / Best regards

Sylvie

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Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]
office: [phone]
LCI website

---- 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<tel:10032>
Phone: [phone]<tel:[phone]>
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