RE: Intracellular Cytokine Staining Methods ??

P.Openshaw Medicine (p.openshaw@ic.ac.uk)
Thu, 1 Jun 1995 10:06:53 +0000 (GMT)

I spent last summer in Anne O'Garra's at DNAX refining methods of cytokine staining of mouse
lymphocytes for flow and we now have several publications coming out. Most
resting cells are negative for IFN gamma, IL-2, IL-4, IL-5 and IL-10 even if
they have seen antigen and are committed to one pattern or another of CK
production. In our protocol, we therefore restimulate with PMA/iononmycin
to induce some synchrony, although different CKs behave very differently in
terms of timing and duration of display. I think this non-specific
restimulation just reveals what committed cells are set to make. In brief:
1) Isolate cells
2) stimulate with PMA/iononmycin or specific antigen/APC
(when compared, both do the same)
3) At various times after stimulation (2-22hrs) add brefeldin A for
the last 2 hrs of culture (to destroy the Golgi and allow
intracellular accumulation)
4) Fix with formaldehyde 2%, 20min RT

Store for up to 2 days or stain straight away
All stain steps are at RT and in permiabilization buffer (PB):
PBS with 1% BSA, 0.5% saponin and azide

1) Treat with PB 10 min
2) Add unconjugated anti CK MAb or isotype control 30 min
(e.g. 11B11 anti IL-4 at 5ug/ml)
3) Wash with PB and add FITC anti-mouse or similar 30 min
4) Block with mouse or rat Ig 0.3 mg/ml
5) Add PE conj anti IFN gamma (AN18 PE) 30 min RT
6) Wash in PBx2 and then wash out saponin
7) Some surface antigens are stable through this protocol and
can be used as 3rd/4th color(s)

This is a modified method is based on that published by Radbruch's group
e.g. Assenmacher Eu. J. Immunol 1994 , 24:1097. If anyone would like our
full protocol, I will gladly send them reprints once papers are accepted
(soon, I hope). Please let me know.

In message Wed, 31 May 1995 19:35:04 +0100,
mhaury@pasteur.fr (Matthias Haury) writes:

> Hello,
>
> Several users in our departement are interested in analysing cytokine
> patterns by FACS. Any methods out there.. sorry I have no access by Mosaic
> to the archive, and I haven't been keeping my eyes open for publications
> in this directions, so I hope I can get a quick overview this way.
>
> Thanks very much of course.
>
> ______________________________________________________________
>
> Matthias Haury _/_/_/ _/_/_/ _/_/_/
> Flowcytometry _/ _/ _/ _/ _/
> Department of Immunology _/ _/_/_/ _/_/_/
> Institut Pasteur _/ _/ _/
> 25 rue du Dr. Roux _/_/_/ _/ _/
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> France
> Tel: 33 1 45 68 85 38
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> Email: mhaury@pasteur.fr
>
*******************************************************************
*Peter Openshaw p.openshaw@ic.ac.uk *
*Respiratory Medicine Tel:+44 171 723 1252 x 5781/6 *
*St. Mary's Hosp. Med. School Fax:+44 171 724 7349 *
*Imperial College, Norfolk Place, London W2 1PG, UK *

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