Re: intracellular IL-4 and IFN-gamma
Julie Auger (jauger@flowcity.bsd.uchicago.edu)
Thu, 30 May 1996 13:28:56 -0500
>Date: Thu, 30 May 1996 10:38:00 -0500
>X-Sender: gvsevent@flowcity.bsd.uchicago.edu
>To: Julie Auger <jauger@flowcity.bsd.uchicago.edu>
>From: gvsevent@flowcity.bsd.uchicago.edu (Gijs A. van Seventer, Ph.D.)
>Subject: Re:
>
>We have performed intracellular staining for cytokines with reagents from
>Pharmingen. We tried various ways of stimulating T cells and the best one
>(and recommended by pharmingen) is stimulation with PMA/ionomycin.
>Unfortunately this stimulatory condition leads to IFN-gamma production even
>in Th2-like cells. Great intercellular staining was observed with this
>activation methods for IFN-gamma, IL-2, TNF-alpha, we failed to detect any
>IL-4, or IL-5 despite the fact that we detect high levels of IL-5 secretion
>under these conditions in our generated Th2-like cells. The bottom line is
>that we are still struggling with this technique and have not found a
>solution to measure IFN-gamma vs. e.g Il-5 in primary cells even after 1 ot
>2 stimulations in vitro. Moreover the most promising activation condition
>induces IFN-gamma in Th2 cells that would not secrete IFN-gamma when
>stimulated through the TCR/CD3 complex. Succes further.
>Gijs van Seventer
>
>
>
>>>X-Sender: brian.rabinovich@mailbox16.utcc.utoronto.ca
>>>Date: Tue, 28 May 1996 03:37:53 -0400
>>>To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
>>>From: brian.rabinovich@utoronto.ca (Brian Rabinovich)
>>>
>>>
>>>Addressed to all those who may be able to aid in the process of staining
>>>for intracellular cytokines using fluorescently tagged mAbs for FACS
>>>analysis on peripheral blood:
>>>
>>>I am currently performing an assay involving intracellular staining of
>>>cytokines from peripheral human blood to assess TH1 and TH2 populations
>>>of CD4+ cells (The protocol I am using is below) . I am currently NOT
>>>stimulating the blood after obtaing it as we wish to assess any possible
>>>alterations in TH1:TH2 between controls and patients with vascular disease
>>>in as pure a fashion as possible. I am finding, however, that unstimulated
>>>CD4+ cells from peripheral blood stain very poorly for both IL-4 and
>>>IFN-gamma, in most cases. I was hoping that someone might have a simple
>>>protocol for stimulating CD4+ cells to synthesize IL-4 or IFN-gamma
>>>depending on their phenotype so that I can stain for a proportionally
>>>amplified cytokine profile. Also, because we are drawing blood from
>>>patients and controls on a regular basis, I am looking for a stimulation
>>>procedure which is as simple and least time consuming as possible. Please
>>>help.....Brian A. Rabinovich, Research Institute, The Wellesley Hospital,
>>>University of Toronto. E MAIL: brian.rabinovich@utoronto.ca
>>>
>>>P.S. Please forward this mesage to anyone else you think could lend a hand
>>>
>>
>>
>>
>
>
>----------------------------------------------------------------------------
>---------
>Gijs van Seventer, Ph.D.
>Assistant Professor
>Department of Pathology
>Committee on Immunology
>University of Chicago
>5841 S. Maryland Avenue
>SBRI, J-541A, MC 1089
>Chicago, IL 60637-1463
>Tel:312-702-9997
>Fax:312-702-3701
>
>
>