An in vitro Co-culture System for the Activation of CD40 by Membrane-presented CD40 Ligand versus Soluble Agonist
| Autor: | Balid Albarbar, Myria Ioannou, Khalidah Ibraheem, Christopher Dunnill, Nikolaos T. Georgopoulos, Albashir Mohamed |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Programmed cell death Cell signaling CD40 biology Chemistry Strategy and Management Mechanical Engineering Metals and Alloys Industrial and Manufacturing Engineering Cell biology Cell membrane 03 medical and health sciences 030104 developmental biology 0302 clinical medicine medicine.anatomical_structure Apoptosis 030220 oncology & carcinogenesis medicine biology.protein Cytokine secretion Receptor Intracellular |
| Zdroj: | BIO-PROTOCOL. 8 |
| ISSN: | 2331-8325 |
| DOI: | 10.21769/bioprotoc.2907 |
| Popis: | One fundamental property of the TNR receptor (TNFR) family relates to how 'signal quality' (the extent of receptor ligation or cross-linking) influences the outcome of receptor ligation, for instance the induction of death in tumour cells. It is unequivocal that membrane-presented ligand (delivered to target cells via cell-surface presentation by co-culture with ligand-expressing third-party cells) induces a greater extent of carcinoma cell death in vitro in comparison to non-cross-linked agonists (agonistic antibodies and/or recombinant ligands). The CD40 receptor epitomises this fundamental property of TNF receptor-ligand interactions, as the extent of CD40 cross-linking dictates cell fate. Membrane-presented CD40 ligand (mCD40L), but not soluble agonists (e.g., agonistic anti-CD40 antibody), induces high level of pro-inflammatory cytokine secretion and causes extensive cell death (apoptosis) in malignant (but not normal) epithelial cells. In this article, we describe a co-culture system for the activation of CD40 by mCD40L and subsequent detection of various features of apoptosis (including cell membrane permeabilisation, DNA fragmentation, caspase activation) as well as detection of intracellular mediators of cell death (including adaptor proteins, pro-apoptotic kinases and reactive oxygen species, ROS). |
| Databáze: | OpenAIRE |
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