Flow cytometry intracellular cytokines

Maino, V.C. and Picker, L.J., 1998. Identification of functional subsets by flow cytometry intracellular detection of cytokine expression, Cytometry, 34, 207, 1998. 

Cytokine/ Flow cytometry intracellular ELISA from cell culture supernatant... 

Intracellular cytokine levels (Flow cytometry using fluorescent labels)... 

Fig. 2. Representative dot plots (PE anti-IL-4 vs. FITC anti-IFN-y) for the analysis of intracellular Th cytokines of CD4+ lymphocytes using the Fastlmmune Cytokine System (BD Pharmingen) by flow cytometry (a) a control subject and (b) an allergic asthmatic patient. The numbers in the quadrants denote the percentages of Thl and Th2 cells (W16). Reproduced with permission from C. K. Wong, C. Y. Ho, F. W. S. Ko, C. H. S. Chan, A. S. S. Ho, D. S. C. Hui, and C. W. K. Lam. Proinflammatory cytokines (IL-17, IL-6, IL-18 and IL-12) and Th cytokines (IFN-y, IL-4, IL-10 and IL-13) in patients with allergic asthma. Clin. Exp. Immunol. 125 177-183, Copyright Blackwell Science Ltd., 2001.

In the 1980s, studies with EAE, an animal model mimicking MS, indicated that interferon y (IFN y) was effective in treating that disease and a trial was initiated to evaluate its potential benefit in human MS. Rather than showing efficacy, in 1987, use of IFNy as a therapy in MS patients caused an increase in clinical exacerbations and forced the clinical trial to terminate early (Panitch et al., 1987). Associated study of IFNy in 20 MS patients indicates increased concentrations of IFNy and TNFa precede the observation of clinical defects (Beck et al., 1988). An evaluation of primary RR-MS patient lymphocytes using flow cytometry supports a correlation between EDSS scores and IFNy secretion (Petereit et al, 2000). Intracellular cytokine immuno-staining of anti CD8-I- T cells reveals a correlation with IFNy and disease phase but not disease activity (Becher et al., 1999). What initially seemed efficacious in the EAE animal model, not only did not decrease MS symptoms but is now felt to be a marker of active inflammatory disease. 

For intracellular cytokine detection by flow cytometry, PBMCs are often used, but to have a more specific analysis, it may be necessary to use cells from other biological fluids (e.g., synovial fluid, cerebrospinal fluid, and bronchoalveo-lar lavage fluid) or to separate cells according to functional characteristics or expression of membrane antigens (e.g., CD3, CD4, CDS, and CD56). 

Godoy-Ramirez K, Franck K, Mahdavifar S, Andersson L, Gaines H. Qptimum culture conditions for specific and nonspecific activation of whole blood and PBMC for intracellular cytokine assessment by flow cytometry. J Immunol Methods 2004 292 1-15. 

Jung T, Schauer U, Heusser C, Neumann C, Rieger C. Detection of intracellular cytokines by flow cytometry. J Immunol Methods 1993 159 197-207. 

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