IL5, intracellular staining

Joanne Lannigan joannelannigan at virginia.edu
Fri Sep 8 16:01:43 EDT 2006


Vladimir:
You don't mention the use of a transport inhibitor. You will need to block
the secretion using a protein transport inhibitor such as Monensin or
BrefeldinA. This would be done during your stimulation. You could also
verify that your cells are permeabilized with an anti-actin fluorescent
antibody.

Joanne Lannigan, MS
Director, Flow Cytometry Core Facility
University of Virginia
Jordan Hall, Room 7067
P.O. Box 800734
Charlottesville, VA 22908-0734
Office: 434-924-0274
Lab: 434-243-2695
Fax: 434-982-1071
email: joannelannigan at virginia.edu

> -----Original Message-----
> From: senyukov.vladimir at hsr.it [mailto:senyukov.vladimir at hsr.it]
> Sent: Friday, September 08, 2006 8:39 AM
> To: Cytometry Mailing List
> Subject: IL5, intracellular staining
> 
> Dear colleagues,
> We have some T clones that produced IL5 as it has been checked up by
> ELISA and ELISPOT. The trouble is I can't catch it by intracellular
> staining. To understand what the problem is I have done a lot of
> preliminary experiments with PBMC. It was two different anti-IL5 Abs
> (APC-rat anti mouse/human, clone TRFK5, and PE-rat anti-human, clone
> JES1-39D10, both from BD Pharmingen) and different activators (PHA, 10
> mkg/ml, PMA, 50 ng/ml) and activation time (4, 18 and 36 hours)  as well
> as different permeability agents (Saponin and TWEEN 20)  that I used but
> without any result. I have had conventional results for IFN-gamma when
> used double cytokine staining but not IL5.  I understand that it should
> be something easy but steel can't understand what it is.
> Any advices would be greatly appreciated
> 
> Vladimir Senyukov, PhD
> Laboratory of Tumor Immunology,
> Cancer Immunotherapy and Gene Therapy program
> DIBIT - San Raffaele Scientific Institute
> Via Olgettina, 58
> 20132 Milano (I)
> tel: +39-02-26434804
> fax: +39-02-26434786
> e-mail: senyukov.vladimir at hsr.it
> 
> 
> 
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