[Cytometry] Protocol conversion to flow

Anna Petrunkina ap542 at cam.ac.uk
Mon Jul 13 05:11:47 EDT 2009


Hi Katie,
methanol is certainly not helpful, especially if your protein is on the 
outer plasma membrane. What did you mean with "very outer plasma membrane" 
Is it localised on the inner layer of the outer membrane or on the cell 
surface? If latter, you do not need permeabilisation at all.

Permeabilization of sperm is very dangerous as the content of the acrosomal 
matrix will leak out and bind proteins from seminal plamsa (see Reprod 
Fertil Dev. 2000;12(7-8):361-71.). Whole procedure will greatly depend on 
the preparation of your samples prior to flow cytometric analysis. Gentle 
fixation may do the trick.
Have a look at the above paper, and if you want to talk about it, e-mail me.

Hope that helps,
Anna


Dr. Anna Petrunkina
Head of Flow Cytometry
Cambridge Institute for Medical Research
University of Cambridge
Wellcome Trust/MRC Building
Addenbrooke's Hospital
Hills Road
Cambridge
CB2 0XY


--On 10 July 2009 11:50 -0400 Katie Hickey <hickeyk at uoguelph.ca> wrote:

>
>
> I work with bovine and porcine sperm cells and am currently attempting to
> convert our slide-based immunofluorescent protein localization protocol
> into a flow (tube-based) protocol.  There are obviously several minor
> issues which we have worked out however we are struggling with cell
> permeabilization methods.  We currently keep a set of cells intact and
> permeabilize the others by dipping the slide in -20 degree methanol for
> 30 seconds.  Methanol exposure in the tube-based assay is much longer and
> is deteriorating our very fragile antibody binding sites. 
>
>
>
> There are many papers suggesting a myriad of permeabilization methods but
> I'm wondering if there is anyone frequently doing this type of conversion
> of protocols that can offer some advice. 
>
>
>
> I've tried triton permeabilization with limited success and am aiming to
> try saponin shortly.  Another consideration is that our protein of
> interest is located in the very outer plasma mambrane of sperm cells,
> which is a fragile and dymanic membrane ... seems that everything
> (temperature, fixation, permeabilization, capacitation status and
> certainly vortexing) drastically affects our ability to visualize the
> protein.
>
>
>
> Thanks in anticipation!
>
>
> Katie
>
> Katie Hickey MSc.
> Research Technician
> Animal and Poultry Science
> ANNU 133
> University of Guelph
> Guelph, ON N1G 2W1
>
> hickeyk at uoguelph.ca
> 519-824-4120 x 58355
>
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