Intracellular markers, flow cytometry

WG Intracellular and cell surface markers (flow cytometry)... 

Flow cytometric determinations of P-selectin and activated GPIIb/llla receptor expression following ADP stimulation have been used to assess platelet inhibition by clopidogrel. Flow cytometry is also a cumbersome method and requires sophisticated instrumentation and well-trained technicians. The phosphorylation state of vasodilator-stimulated phos-phoprotein (VASP) is a specific intracellular marker of clopidogrel-induced P2Y 2 receptor inhibition and can also be measured by flow cytometry. Permeation of the membrane and the use of monoclonal antibodies specific for phosphory-lated VASP are required in this method (15,88). 

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. 

Cell activation can lead to differentiation of certain cells into more mature cells that exhibit different structural and functional form. For example, immature hematopoietic cells can be stimulated by some proteins to mature, by changing cell morphology and by inducing expression of different intracellular and surface markers. Expression of differentiation markers can be detected and monitored by flow cytometry using specific antibodies against cell surface or intracellular markers. 

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