From: Calman Prussin (CPRUSSIN@atlas.niaid.nih.gov)
Date: Thu Feb 05 1998 - 09:06:00 EST
In an early paper we performed a head to head comparison between ELISA and cytokine flow in 5 subjects and found a statistically significant correlation for IL-4 and IL-5 and a reasonable trend for IFN-g (J.I. 154, 4294-4301, 1995). The data is noted, but there is no figure. There was less intrasubject variability for IFN and hence it was more difficult to make a nice curve with the few subjects we studied. Although I'm sure one can come up with scenarios that demonstrate a disconnect between ELISA and cytokine flow, the overwhelming consensus is that there is a correlation. I would disagree with the remarks below "you can't distinguish cytokine that is produced by the cell from cytokine that is internalized by the cell" and then goes on to cite my data for support. We found, as have Assenmacher and Radbruch, a population of cells that stained EXTRACELLULARLY for cytokine. However, this staining was of a lower frequency and fluorescence intensity and most importantly, was only found on cells that also stained for intracellular cytokine. We found no evidence of cells passively internalizing cytokine. Furthermore, although production does not equal secretion, in my experience T cells are very efficient at secreting cytokine and hence what stains intracellularly generally does reflect what is secreted. Certainly users should be aware of these potential limitations, but the published data do not support them being considered as frequent problems which invalidate the technique. > _______________________ > Calman Prussin > Laboratory of Allergic Diseases > NIAID/ National Institutes of Health > > ---------- > From: david.mcfarland@mcmail.vanderbilt.edu > Sent: Monday, February 2, 1998 18:08 > To: Cytometry Mailing List > Subject: Intracellular cytokine staining versus secreted cytokine > > > Try these: > > Jung T, Schauer U, Heusser C, Neumann C, Rieger C: Detection of > intracellular > cytokines by flow cytometry. J. Immunol. Met. 150:197-207, 1993. > > Kabilan L, Anderson G, Lolli F, Ekre H-P, Olsson T, Troye-Blomberg M: > Detection > of intracellular expression and secretion of interferon-gamma at the > single-cell > level after activation of human T cells with tetanus toxoid in vitro. > Eur. J. > Immunol. 20:1085-1089,1990. > > One problem with intracellular staining seems to be the fact that you > can't > distinguish cytokine that is produced by the cell from cytokine that > is > internalized by the cell. In addition, just because a cell is a high > producer, > that doesn't necessarily mean it is a high secretor. Prussin and > Metcalfe have > also shown that there is an "extracellular pool" of cytokine that can > be > labelled without permeablising the cells. See: > > Prussin C and Metcalfe DD: Detection of intracytoplasmic cytokine > using flow > cytometry and directly conjugated anti-cytokine antibodies. J. > Immunol. Met. > 188:117-128, 1995. > > Happy Flowing! > > > David McFarland > Howard Hughes Medical Institute > Flow Cytometry Facility > Vanderbilt University Medical Center > > ______________________________________________________________________ > _________ > Subject: Intracellular cytokine staining versus secreted cytokine > From: Margaret Cooley <m.cooley@unsw.EDU.AU> at +inet > Date: 02/02/1998 2:24 PM > > > Hi all > > Can anyone point me to references where intracellular cytokine > staining by > flow cytometry was compared directly to assays (e.g. ELISA) of > secreted > cytokine or of cytokine message? What I am interested in is whether > there > is any data correlating frequencies of cytokine producing cells with > total > secreted cytokine or cytokine mRNA levels. I know work relating mRNA > levels to secreted cytokine, but not intracellular cytokine to > secreted > cytokine > > Thanks for your help. > > Margaret A. Cooley > School of Microbiology and Immunology > The University of New South Wales > Sydney, NSW 2052 Australia > Phone:61-2-9385-3761 > Fax: 61-2-9385-1591 > > >
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