Attaching B-cells to a coverslip

[Michal Jarnik]

A question:

A user needs to attach B-cells to a coverslip for further
immunofluorescent labeling. Although glass is coated with Poly-Lys, the
cells do wash away during the labeling and washing steps. Any smart
ideas what to use to cover the coverslips and/or how to additionally
treat them upon attachment of the cells?

Thanks,

Michal

--

Michal Jarnik, Ph.D.
Cell Imaging Facility
Electron Microscope Facility
Fox Chase Cancer Center

[Sam's Mail]

One choice we have used successfully for B-cells is Cell-Tak from BD Biosciences.
--
Samuel A. Connell
Director of Light Microscopy
Cell & Tissue Imaging Center
St. Jude Children's Research Hospital
262 Danny Thomas Place
Memphis, TN 38105-3678
Office [phone]
Cell [phone]
[hidden email]



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Michal Jarnik
Sent: Wednesday, March 17, 2010 1:44 PM
To: [hidden email]
Subject: Attaching B-cells to a coverslip. .

A question:

A user needs to attach B-cells to a coverslip for further
immunofluorescent labeling. Although glass is coated with Poly-Lys, the
cells do wash away during the labeling and washing steps. Any smart
ideas what to use to cover the coverslips and/or how to additionally
treat them upon attachment of the cells?

Thanks,

Michal

--

Michal Jarnik, Ph.D.
Cell Imaging Facility
Electron Microscope Facility
Fox Chase Cancer Center


Email Disclaimer:  www.stjude.org/emaildisclaimer

[Andrea Pfeifer]

Michal,

what you could also do is fix and stain the cells still in suspension in an
Eppendorf tube as you would do for flow cytometry and remove the last wash
step as completely as possible, then resuspend the cells in your embedding
medium and place them on your slide. This worked well for me with primary
erythroid cells (I would probably not try this with a methanol fixation,
have only done it with PFA).

Best
Andrea

[mmodel]

Sorry, I missed the beginning of the discussion, but depending on your applications, fixed suspended cells can be attached to glass by simply transferring them to water and letting them dry on the coverslip. They will become a little flatter from drying, and lipids may get messed up, but for many applications this should work fine.

Michael Model, Ph.D.
Confocal Microscopy,
Dpt Biological Sciences,
1275 University Esplanade,
Kent State University, Kent, OH 44242
tel. [phone]


________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Andrea Pfeifer [[hidden email]]
Sent: Thursday, March 18, 2010 3:22 AM
To: [hidden email]
Subject: Re: Attaching B-cells to a coverslip

Michal,

what you could also do is fix and stain the cells still in suspension in an
Eppendorf tube as you would do for flow cytometry and remove the last wash
step as completely as possible, then resuspend the cells in your embedding
medium and place them on your slide. This worked well for me with primary
erythroid cells (I would probably not try this with a methanol fixation,
have only done it with PFA).

Best
Andrea

[Andreas Bruckbauer]

This can depend on the B cells, try  fibronectin, see the current (Feb 2010) issue of Immunity, page 187
Andreas

-----Original Message-----
From: MODEL, MICHAEL <[hidden email]>
To: [hidden email]
Sent: Thu, 18 Mar 2010 10:31
Subject: Re: Attaching B-cells to a coverslip

Sorry, I missed the beginning of the discussion, but depending on your 

applications, fixed suspended cells can be attached to glass by simply 

transferring them to water and letting them dry on the coverslip. They will 

become a little flatter from drying, and lipids may get messed up, but for many 

applications this should work fine.



Michael Model, Ph.D.

Confocal Microscopy,

Dpt Biological Sciences,

1275 University Esplanade,

Kent State University, Kent, OH 44242

tel. [phone]





________________________________________

From: Confocal Microscopy List [[hidden email]] On Behalf Of 

Andrea Pfeifer [[hidden email]]

Sent: Thursday, March 18, 2010 3:22 AM

To: [hidden email]

Subject: Re: Attaching B-cells to a coverslip



Michal,



what you could also do is fix and stain the cells still in suspension in an

Eppendorf tube as you would do for flow cytometry and remove the last wash

step as completely as possible, then resuspend the cells in your embedding

medium and place them on your slide. This worked well for me with primary

erythroid cells (I would probably not try this with a methanol fixation,

have only done it with PFA).



Best

Andrea=

[Steffen Dietzel]

Michal,

we used poly-L-Lysin successfully to get
peripheral blood cells on the coverslip, which
should contain a good deal of B-cells. Maybe it
depends on the details of the protocoll? We used
150000 Da poly-L-Lysin. The details can be found
e.g. in Hepperger et al, Chromosoma Volume 116, Number 2 / April 2007,  here:
http://www.springerlink.com/content/u16256nu17287508/?p=c1f4277947524f12a76bc26ff4a24998&pi=3
In this study we used MEL cells transgenic for a
GFP-labeled chromosome region, to better compare
preservation during various fixation procedures,
but the protocoll is the same for lymphocytes. The article is open access.

Steffen



At 19:44 17.03.2010, you wrote:

>A question:
>
>A user needs to attach B-cells to a coverslip
>for further immunofluorescent labeling. Although
>glass is coated with Poly-Lys, the cells do wash
>away during the labeling and washing steps. Any
>smart ideas what to use to cover the coverslips
>and/or how to additionally treat them upon attachment of the cells?
>
>Thanks,
>
>Michal
>
>--
>
>Michal Jarnik, Ph.D.
>Cell Imaging Facility
>Electron Microscope Facility
>Fox Chase Cancer Center

--
---------------------------------------------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)