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

[TSwayne]

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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: [phone]
[hidden email]<mailto:[hidden email]>

[Tim Feinstein]

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Hi Theresa,

Have you thought about polarization?  A plasma membrane protein would have greater anisotropy than a cytosolic protein since it is tumbling much more slowly.  It should be easy to have membrane/cytosol controls to calibrate the experiment.  

Best,


Tim

Timothy Feinstein, Ph.D.
Research Scientist
University of Pittsburgh Department of Developmental Biology


On 3/6/17, 2:47 PM, "Confocal Microscopy List on behalf of Swayne, Theresa C." <[hidden email] on behalf of [hidden email]> 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<https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fhiccc.columbia.edu%2Fresearch%2Fsharedresources%2Fconfocal&data=01%7C01%7Ctnf8%40PITT.EDU%7C409a6144ee024a97aa4508d464c9bdbd%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1&sdata=CaG20A3kRW9Sz8U5NCFoOlGHxpLiB2LZDsmHb1FGVVI%3D&reserved=0>
   
    Herbert Irving Comprehensive Cancer Center
    Columbia University Medical Center
    1130 St. Nicholas Ave., Room 222A
    New York, NY 10032
    Phone: [phone]
    [hidden email]<mailto:[hidden email]>
   
   

[George McNamara]

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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.pdf

Probably too ambitious for today, but I encourage you to consider
expanding your T-cells with Expansion Microscopy,

http://expansionmicroscopy.org

http://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:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
>
> 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: [phone]
> [hidden email]<mailto:[hidden email]>
>

--


George McNamara, PhD
Houston, TX 77054
[hidden email]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75/
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650

[Renato A. Mortara]

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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.


------------------------------------
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]
[hidden email]<mailto:[hidden email]>

[mmodel]

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Or maybe (I am combining Renato's and George's suggestions) with both anti-epitope and anti-CD4 and look for FRET

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Renato
Sent: Monday, March 6, 2017 3:15 PM
To: [hidden email]
Subject: RES: How to differentiate plasma membrane vs. cytoplasm in Jurkat lymphocytes?

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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.


------------------------------------
Theresa Swayne, Ph.D.
Manager
Confocal and Specialized Microscopy Shared Resource<https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fhiccc.columbia.edu%2Fresearch%2Fsharedresources%2Fconfocal&data=01%7C01%7Cmmodel%40KENT.EDU%7C173ee62949ce477c969f08d464cdb832%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1&sdata=uiGYhq3r72NM1%2FnLXQQlClD0u8M3y%2FR%2F2%2FD3xBiGbWM%3D&reserved=0>

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

[Tim Feinstein]

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If you have to work with fixed cells, TIRF might be a good way to go.  Cytosolic proteins would get brighter as you adjust the TIRF angle from more stringency to less while PM proteins would not.  If you are working with live cells, FRAP or FLIP would tell you whether they diffuse like membrane-bound proteins or soluble proteins.  Membrane mobility would go down significantly if you decrease the temperature a little.  

Tim
       
Timothy Feinstein, Ph.D.
Research Scientist
University of Pittsburgh Department of Developmental Biology


On 3/6/17, 2:47 PM, "Confocal Microscopy List on behalf of Swayne, Theresa C." <[hidden email] on behalf of [hidden email]> wrote:

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    Post images on https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=01%7C01%7Ctnf8%40PITT.EDU%7C409a6144ee024a97aa4508d464c9bdbd%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1&sdata=whJpk0Px%2B7ODn9%2BNN6my6T2k%2FNfz01wf2DoKWAAhqDo%3D&reserved=0 and include the link in your posting.
    *****
   
   
    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<https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fhiccc.columbia.edu%2Fresearch%2Fsharedresources%2Fconfocal&data=01%7C01%7Ctnf8%40PITT.EDU%7C409a6144ee024a97aa4508d464c9bdbd%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1&sdata=CaG20A3kRW9Sz8U5NCFoOlGHxpLiB2LZDsmHb1FGVVI%3D&reserved=0>
   
    Herbert Irving Comprehensive Cancer Center
    Columbia University Medical Center
    1130 St. Nicholas Ave., Room 222A
    New York, NY 10032
    Phone: [phone]
    [hidden email]<mailto:[hidden email]>
   
   

[Artem Pliss]

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The cytosolic  fraction of a protein can be differentiated from the
membrane fraction by FLIM. Membrane-bound fluorophore will have a shorter
lifetime; this is based on difference between refractive indices of cytosol
and plasma membrane.  See for reference "Refractive index sensing of green
fluorescent proteins in living cells using fluorescence lifetime imaging
microscopy" Biophysical Journal (2008), 94 (8), L67-L69
Best regards

On Mon, Mar 6, 2017 at 3:31 PM, Feinstein, Timothy N <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> If you have to work with fixed cells, TIRF might be a good way to go.
> Cytosolic proteins would get brighter as you adjust the TIRF angle from
> more stringency to less while PM proteins would not.  If you are working
> with live cells, FRAP or FLIP would tell you whether they diffuse like
> membrane-bound proteins or soluble proteins.  Membrane mobility would go
> down significantly if you decrease the temperature a little.
>
> Tim
>
> Timothy Feinstein, Ph.D.
> Research Scientist
> University of Pittsburgh Department of Developmental Biology
>
>
> On 3/6/17, 2:47 PM, "Confocal Microscopy List on behalf of Swayne, Theresa
> C." <[hidden email] on behalf of [hidden email]>
> wrote:
>
>     *****
>     To join, leave or search the confocal microscopy listserv, go to:
>     https://na01.safelinks.protection.outlook.com/?url=
> http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%
> 3Dconfocalmicroscopy&data=01%7C01%7Ctnf8%40PITT.EDU%
> 7C409a6144ee024a97aa4508d464c9bdbd%7C9ef9f489e0a04eeb87cc3a526112
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>     Post images on https://na01.safelinks.protection.outlook.com/?url=
> http%3A%2F%2Fwww.imgur.com&data=01%7C01%7Ctnf8%40PITT.EDU%
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> reserved=0 and include the link in your posting.
>     *****
>
>
>     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<https://na01.
> safelinks.protection.outlook.com/?url=http%3A%2F%2Fhiccc.
> columbia.edu%2Fresearch%2Fsharedresources%2Fconfocal&
> data=01%7C01%7Ctnf8%40PITT.EDU%7C409a6144ee024a97aa4508d464c9bdbd%
> 7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1&sdata=
> CaG20A3kRW9Sz8U5NCFoOlGHxpLiB2LZDsmHb1FGVVI%3D&reserved=0>
>
>     Herbert Irving Comprehensive Cancer Center
>     Columbia University Medical Center
>     1130 St. Nicholas Ave., Room 222A
>     New York, NY 10032
>     Phone: [phone]
>     [hidden email]<mailto:[hidden email]>
>
>
>
>

--
Artem Pliss
Research Assistant Professor
432 Natural Sciences Complex
Institute for Lasers, Photonics and Biophotonics
State University of New York, University at Buffalo
Buffalo, NY 14260 - 3000
Ph.: [phone]
Fax: [phone]

[Mike Ignatius-3]

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I worked in lab at UCSF that tried to do LM based assignment of a membrane
associated protein.  They published that it was external and a much sought
after ECM receptor.  Turned out it was internal and the retraction was
forthcoming and quite embarrassing.   Many other examples of this.  IMHO it
takes EM to definitively make that assignment.



On Mon, Mar 6, 2017 at 1:26 PM, Artem Pliss <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> The cytosolic  fraction of a protein can be differentiated from the
> membrane fraction by FLIM. Membrane-bound fluorophore will have a shorter
> lifetime; this is based on difference between refractive indices of cytosol
> and plasma membrane.  See for reference "Refractive index sensing of green
> fluorescent proteins in living cells using fluorescence lifetime imaging
> microscopy" Biophysical Journal (2008), 94 (8), L67-L69
> Best regards
>
> On Mon, Mar 6, 2017 at 3:31 PM, Feinstein, Timothy N <[hidden email]>
> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > If you have to work with fixed cells, TIRF might be a good way to go.
> > Cytosolic proteins would get brighter as you adjust the TIRF angle from
> > more stringency to less while PM proteins would not.  If you are working
> > with live cells, FRAP or FLIP would tell you whether they diffuse like
> > membrane-bound proteins or soluble proteins.  Membrane mobility would go
> > down significantly if you decrease the temperature a little.
> >
> > Tim
> >
> > Timothy Feinstein, Ph.D.
> > Research Scientist
> > University of Pittsburgh Department of Developmental Biology
> >
> >
> > On 3/6/17, 2:47 PM, "Confocal Microscopy List on behalf of Swayne,
> Theresa
> > C." <[hidden email] on behalf of
> [hidden email]>
> > wrote:
> >
> >     *****
> >     To join, leave or search the confocal microscopy listserv, go to:
> >     https://na01.safelinks.protection.outlook.com/?url=
> > http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%
> > 3Dconfocalmicroscopy&data=01%7C01%7Ctnf8%40PITT.EDU%
> > 7C409a6144ee024a97aa4508d464c9bdbd%7C9ef9f489e0a04eeb87cc3a526112
> > fd0d%7C1&sdata=Do6lnQHtibW9oSyl4MkRNFIPaRUYQ1
> %2Bfr21El53AunU%3D&reserved=0
> >     Post images on https://na01.safelinks.protection.outlook.com/?url=
> > http%3A%2F%2Fwww.imgur.com&data=01%7C01%7Ctnf8%40PITT.EDU%
> > 7C409a6144ee024a97aa4508d464c9bdbd%7C9ef9f489e0a04eeb87cc3a526112
> > fd0d%7C1&sdata=whJpk0Px%2B7ODn9%2BNN6my6T2k%2FNfz01wf2DoKWAAhqDo%3D&
> > reserved=0 and include the link in your posting.
> >     *****
> >
> >
> >     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<https://na01.
> > safelinks.protection.outlook.com/?url=http%3A%2F%2Fhiccc.
> > columbia.edu%2Fresearch%2Fsharedresources%2Fconfocal&
> > data=01%7C01%7Ctnf8%40PITT.EDU%7C409a6144ee024a97aa4508d464c9bdbd%
> > 7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1&sdata=
> > CaG20A3kRW9Sz8U5NCFoOlGHxpLiB2LZDsmHb1FGVVI%3D&reserved=0>
> >
> >     Herbert Irving Comprehensive Cancer Center
> >     Columbia University Medical Center
> >     1130 St. Nicholas Ave., Room 222A
> >     New York, NY 10032
> >     Phone: [phone]
> >     [hidden email]<mailto:[hidden email]>
> >
> >
> >
> >
>
>
> --
> Artem Pliss
> Research Assistant Professor
> 432 Natural Sciences Complex
> Institute for Lasers, Photonics and Biophotonics
> State University of New York, University at Buffalo
> Buffalo, NY 14260 - 3000
> Ph.: [phone]
> Fax: [phone]
>

--
Ignatius Family
225 East 39th Ave
Eugene OR 97405
541 484 1286
[hidden email]

[Sylvie Le Guyader]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

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

@@@@@@@@@@@@@@@@@@@@@@@@

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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To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****


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]>
[hidden email]<mailto:[hidden email]><mailto:[hidden email]<mailto:[hidden email]>>

[darren.thomson]

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

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?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****


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: [phone]
[hidden email]<mailto:[hidden email]>

[TSwayne]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****


Dear confocalists,

I am grateful for all of the excellent suggestions!

I don’t have easy access to a fluorescence polarization or FLIM system, though there are facilities nearby. The protein is not exposed to the extracellular milieu, so I wouldn’t be able to label it that way.

However, we could certainly try the ideas for TIRF, FRAP, “squeezing,” and simply maximizing resolution in confocal. But I also see that first I need to clarify the possibilities for localization, because if the protein is expected to accumulate near the PM it will be a very different problem to distinguish membrane-attached from non-attached.

To summarize the strategies that were recommended, roughly in reverse chronological order:

1) CellMask stays on membrane for up to ~40 min in macrophages, but is more internalized after fixation. (Darren Thomson)

2) If the protein is cytoplasmic but accumulated near the membrane, SIM or other techniques would be required to determine the localization. If it is distributed throughout the cytoplasm, you could use Brilliant Violet or similar antibody for protein of interest and controls; use a green cytoplasmic marker. Image cells with highest NA, small pinhole, and coverslip correction, at equator, and look for cells with > 400 nm width (at least 4 pixels) of visible cytoplasm. Compare the violet intensity in the half of cytoplasm closer to the membrane versus the half near the nucleus. (Sylvie Le Guyader)

3)   It’s too risky to assess external vs. internal via LM and EM should be used. (Mike Ignatius)

4) An advanced profile analysis could be used similar to that employed by the Ewa Paluch lab (http://dx.doi.org/10.1016/j.bpj.2013.05.057) who determined actin cortex localization and thickness with precision better than the diffraction limit. (Christian Eggeling)

5) FLIM based on refractive index differences in membrane vs. cytosol, as done by the Cees Otto lab (http://dx.doi.org/10.1529/biophysj.107.127837) (Artem Pliss)

6) For fixed cells, TIRF at a series of angles (cytosolic proteins would get brighter as you go deeper); for live cells, FRAP/FLIP, looking for greater mobility of soluble proteins. (Tim Feinstein)

7) Make resolving the cytoplasm easier by changing cell morphology: compression, activation, or expansion microscopy. (George McNamara)

8) Fluorescence polarization, looking for greater anisotropy in cytosolic proteins (Tim Feinstein)

9) Identify surface-exposed epitopes by labeling live cells on ice. (Renato Mortara)


Thank you again. I love this community!


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

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com<http://www.imgur.com/> and include the link in your posting.
*****


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: [phone]
[hidden email]<mailto:[hidden email]>

[George McNamara]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi again Theresa,

The Human Protein Atlas team has a list of 30 subcellular sites: so your
favorite epitope tagged protein will likely be in one or more of those
locations (and commercially available antibodies), and just had a
webinar (~60 minutes, a PDF slide deck is available for download once
registered)

http://www.proteinatlas.org/humancell

http://www.sciencemag.org/custom-publishing/webinars/high-resolution-look-human-cell-introducing-human-cell-atlas

Their "cell atlas" is not based on human T-cells, so I still suggest
prioritizing with respect to Singeton and Wulfing, re:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2694444/table/T1

Feel free to mention the Confocal Listserv in your publication(s)
acknowledgements.

best wishes,

George


On 3/9/2017 10:12 AM, Swayne, Theresa C. wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
>
> Dear confocalists,
>
> I am grateful for all of the excellent suggestions!
>
> I don’t have easy access to a fluorescence polarization or FLIM system, though there are facilities nearby. The protein is not exposed to the extracellular milieu, so I wouldn’t be able to label it that way.
>
> However, we could certainly try the ideas for TIRF, FRAP, “squeezing,” and simply maximizing resolution in confocal. But I also see that first I need to clarify the possibilities for localization, because if the protein is expected to accumulate near the PM it will be a very different problem to distinguish membrane-attached from non-attached.
>
> To summarize the strategies that were recommended, roughly in reverse chronological order:
>
> 1) CellMask stays on membrane for up to ~40 min in macrophages, but is more internalized after fixation. (Darren Thomson)
>
> 2) If the protein is cytoplasmic but accumulated near the membrane, SIM or other techniques would be required to determine the localization. If it is distributed throughout the cytoplasm, you could use Brilliant Violet or similar antibody for protein of interest and controls; use a green cytoplasmic marker. Image cells with highest NA, small pinhole, and coverslip correction, at equator, and look for cells with > 400 nm width (at least 4 pixels) of visible cytoplasm. Compare the violet intensity in the half of cytoplasm closer to the membrane versus the half near the nucleus. (Sylvie Le Guyader)
>
> 3)   It’s too risky to assess external vs. internal via LM and EM should be used. (Mike Ignatius)
>
> 4) An advanced profile analysis could be used similar to that employed by the Ewa Paluch lab (http://dx.doi.org/10.1016/j.bpj.2013.05.057) who determined actin cortex localization and thickness with precision better than the diffraction limit. (Christian Eggeling)
>
> 5) FLIM based on refractive index differences in membrane vs. cytosol, as done by the Cees Otto lab (http://dx.doi.org/10.1529/biophysj.107.127837) (Artem Pliss)
>
> 6) For fixed cells, TIRF at a series of angles (cytosolic proteins would get brighter as you go deeper); for live cells, FRAP/FLIP, looking for greater mobility of soluble proteins. (Tim Feinstein)
>
> 7) Make resolving the cytoplasm easier by changing cell morphology: compression, activation, or expansion microscopy. (George McNamara)
>
> 8) Fluorescence polarization, looking for greater anisotropy in cytosolic proteins (Tim Feinstein)
>
> 9) Identify surface-exposed epitopes by labeling live cells on ice. (Renato Mortara)
>
>
> Thank you again. I love this community!
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Swayne, Theresa C.
> Sent: 06 March 2017 19:48
> To: [hidden email]<mailto:[hidden email]>
> Subject: How to differentiate plasma membrane vs. cytoplasm in Jurkat lymphocytes?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com<http://www.imgur.com/> and include the link in your posting.
> *****
>
>
> 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: [phone]
> [hidden email]<mailto:[hidden email]>
>

--


George McNamara, PhD
Houston, TX 77054
[hidden email]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75/
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650