Eosinophil Intracellular Signalling: Apoptosis
Eosinophil apoptosis is considered critical for the resolution of eosinophilic inflammation in the airways of asthmatics. Apoptosis can be mediated by an extrinsic receptor-activated pathway or alternatively by an intrinsic pathway via distortion of mitochondrial function. Both of these pathways lead to activation of the caspase cascade resulting in degradation of cellular components. We describe here two methods to explore intracellular mechanisms mediating eosinophil apoptosis. Eosinophil staining by fluorescent probe JC-1 followed by flow cytometric analysis is a reliable method for determination of the state of mitochondrial membrane potential (∆Ψm). Lost ∆Ψm indicates distorted mitochondrial function and apoptosis. We also describe a method to explore the activation of effector caspase-6 by assessing degradation of its substrate lamin A/C by immunoblotting.
Key wordsEosinophils Apoptosis Mitochondrial membrane potential JC-1 Caspase-6 Lamin A/C Immunoblotting
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