attachment of primary T cells

[Gabriela Gorelik]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear all,
 
I need to attach primary T cells from peripheral blood to study translocation of proteins by confocal microscopy or delta vision microscope. I couldn't get the cells attached to the surface, they moved constantly and therefore I cannot get the images.
I worked with glass bottom culture dishes poly-d-lysine coated but didn't work.
I will appreciate any advice you can give me to attach primary T cells.
Thank you,
 
Gabriela
**********************************************************
Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues

[Josef Gotzmann]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear Gabriela,

In my hands the "adhesion slides"
(http://www.marienfeld-superior.com//index.php/microscope-slides/articles/adhesion-slides.html)

have always been working nicely for hematopoietic cells - far better than
any coating or centrifugation. Easy to use and can also be used for
cultivating cells.

Hope this helps,
Josef

[ncobb]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

My colleague had a similar challenge with lightly attaching human
neutrophils to glass surfaces. He would isolate neutrophils from blood and
suspend in HBSS with HEPES. He would supplement the buffer with fetal bovine
serum (FBS). 4% serum was high enough that the neutrophils would not stick
to any glass surface including glass micropipettes. Approximately 0.5% FBS
would allow the neutrophils to lightly attach to a glass surface, but not
spread. Without serum, neutrophils and lymphocytes will strongly adhere and
begin spreading and crawling. Poly-L-Lysine will normally increase the
likelihood of a strong adhesion, but with a high enough concentration of
serum proteins the charged sites will be passivated  before the cell comes
in contact with the surface. If you'd like to try a Silicon or Silicon
Nitride membrane I'd be happy to send you a sample.

[Gabriela Gorelik]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Josef
 
Thank you so much for your information. I will try those slides.
 
Gabriela

>>> On 2/15/2011 at  9:48 AM, in message <LISTSERV%[hidden email]>, Josef Gotzmann <[hidden email]> wrote:
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy 
*****

Dear Gabriela,

In my hands the "adhesion slides"
(http://www.marienfeld-superior.com//index.php/microscope-slides/articles/adhesion-slides.html)

have always been working nicely for hematopoietic cells - far better than
any coating or centrifugation. Easy to use and can also be used for
cultivating cells.

Hope this helps,
Josef
**********************************************************
Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues

[Amanda M. Lawrence]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

The annual meeting of the Southeastern Microscopy Society (SEMS) will be held May 18-20 in Decatur, Georgia, just east of Atlanta.  We will have a day and a half of presentations, and this year we are privileged to have as invited speakers Drs. Paul Hvala, Daniela Nicastro, Amelia Kempere, and Michael Oliveri.  Students are encouraged to submit an abstract for the Ruska Award.  There is also the opportunity to participate in a pre-meeting workshop on plunge freezing  and cryo-TEM at Emory University, a Vendors' Social on May 18, and a banquet on May 19.  Visit us at www.southeasternmicroscopy.org<http://www.southeasternmicroscopy.org> for details. Please come and join us!

All microscopists in the southeast region, irrespective of discipline, are invited and encouraged to join the Southeast Microscopy Society. SEMS is one of the most active Local Affiliate Societies of the Microscopy Society of America and an excellent venue for students, technologists and faculty to share their data with and to meet other scientists from our region with similar interests.

[SUBSCRIBE CONFOCALMICROSCOPY Estelle Chiari]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

We tried the BD cell tak product, cells are very well attached but we  have
trouble to figure out if the picture fussiness (DAPI is horrendous) are due
do the mounting media reacting with the cell tak or if cell tak isr eally
not good for confocal...

[Boswell, Carl A - (cboswell)]

Hello whomever you are,

Cell Tak is essentially a glue.  If you don't block the exposed material, most anything will stick, including dyes.  Do a blocking incubation like you would for immunostaining or Western blot.  Also, it pays to remember that this material, much like other adhesive coatings, may alter the natural morphology and physiology of cells because 1) they are cemented to the surface and cannot move as freely as in vivo and 2) surface receptors are not interacting with  natural substrates.  
C


Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
Univ. of Arizona
520-954-7053
FAX [phone]


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of SUBSCRIBE CONFOCALMICROSCOPY Estelle Chiari
Sent: Wednesday, February 16, 2011 7:06 AM
To: [hidden email]
Subject: Re: attachment of primary T cells

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

We tried the BD cell tak product, cells are very well attached but we  have trouble to figure out if the picture fussiness (DAPI is horrendous) are due do the mounting media reacting with the cell tak or if cell tak isr eally not good for confocal...