One inherent problem of cytokine measurement by flow is the time point chosen for the analysis. For example, the relative frequency of IL-4 and IFN-g producers will change if the same population of cells is fixed and stained 6 hour after in vitro restimulation or 16 hour after restimulation. IL-4 + cells tend to be present earlier during the activation than IFN-g+ cells. IFN-g positive cells on the other hand accumulate over the time of activation. Therefore for each of the cytokines that one is interested in, a good knowledge on the kinetics of production are important. This means that measuring both cytokines at let's say 6 hours will lead to an overestimation of the relative frequency of IL-4 : IFNg producers. ELISA should have less of a problem with these differences in the kinetics, as it measures accumulated product. However, IL-4 can be taken up (like IL-2) by the T cells and a long culture might therefore underestimate the amounts produced. That of course leads to the question of what one wants to measure. Restimulation in vitro, independent on what the method of read-out is can only determine the POTENTIAL of these cells, not what they actually do in vivo. To make things more complicated, 16hour stimulation in the presence of either monensin or brefeldinA (golgi-blocker to allow the accumulation of cytokines for detection intracytoplamically) will induce cell death. I don't know whether the death of the cells affect certain subpopulations differentially and therefore long-term stimulation for flow might scew the analysis by inducing death in certain subsets. It must also taken into consideration that cells might increase their production of a certain cytokine i.e. the same number of cells produces a higher amount of cytokine, obviously that is not something that one could determine by flow. As transcription of cytokine genes can occur from only one allele or from both alleles, I think this could clearly be a way of regulating overall cytokine production in vivo. A last point i would like to make is that although flow has the advantage of studying simultaneously the phenotype and the cytokine production profile ofvarious cells and cell subsets in a mixed population, it is not known whether cell-cell interactions between these subpopulations regulate their differential expression. Therefore at the end of the day cell subpopulations have to be isolated cleanly to determine their in vitro potential. In summary, if flow analysis is chosen as a read-out for cytokine production than I would strongly recommend to do a extensive kinetic analysis on the cytokines of interest, i.e stimulating cell populations for different times and adding the golgi-blockers always only for the last 4-6 hours of culture. I hope this makes sense, Nicole Nicole Baumgarth Stanford University School of Medicine Dept Genetics 300 Pasteur Drive, Beckman Center B007 Stanford, California 94305 - 5318 FAX: 650 - 725 8564 Phone: 650 - 723 7149
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