This volume provides the most commonly used methods and protocols to study the apoptotic and non-apoptotic roles of CD95. Chapters explore molecular, biochemical, cellular methods and animal models to in order to better understand the biological functions of this cytokine. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, CD95: Methods and Protocols aims to foster original research on CD95/CD95L couple.
Table of Contents
Production of the Non-apoptotic Metalloprotease-cleaved CD95L and its Cytotoxic Recombinant Counterpart Designed Ig-CD95L.- CD95 Stimulation with CD95L and DISC Analysis.- Immunoprecipitation of Death Inducing Signaling Complex by Caspase-8.- In vitro Evaluation of the Apoptosis Function in Human Activated T cells.- Proximity Ligation Assay (PLA) to Evaluate DISC and MISC Composition.- Fluorometric Methods for Detection of Mitochondrial Membrane Depolarization.- Generation and Application of Bioluminescent CD95 Ligand Fusion Proteins.- CD95-mediated Calcium Signaling.- CD95-mediated Proton Regulation.- Study of the CD95-mediated Non-apoptotic Signaling Pathway – PI3K.- Organelle Separation and Cell Signaling.- Boyden Chamber Assay to Study of Cell Migration Induced by Metalloprotease Cleaved-CD95L.- Isolation of Lipid Rafts through Discontinuous Sucrose Gradient Centrifugation and Fas/CD95 Death Receptor Localization in Raft Fractions.- Quantifying CD95/cl-CD95L Implications in Cell Mechanics And Membrane Tension by Atomic Force Microscopy Based Force Measurements.- Sketching of CD95 Oligomers by In Silico Investigations.- Site-specific Detection of Tyrosine Phosphorylated CD95 Following Protein Separation by Conventional and Phospho-Protein Affinity SDS-PAGE.- Detection of S-acylated CD95 by Acyl-biotin Exchange.- Exploration of Fas S-nitrosylation by the Biotin Switch Assay.- Method to Measure Sphingomyelin Synthase Activity Changes in Response to CD95L.- Liquid Chromatography-High Resolution Mass Spectrometry Method to Study Sphingolipid Metabolism Changes in Response to CD95L.- CD95 and the MRL-lpr Mouse Model.