Dectin-mediated Ca2+ flux in dendritic cells.

• Main Author: Ismawati Mohamad Amin.
• Other Authors: Ruedl Christiane
• Format: Final Year Project
• Language: English
• Published: 2010
• Subjects: DRNTU::Science::Biological sciences::Microbiology::Immunology
• Online Access: http://hdl.handle.net/10356/39922
• Institution: Nanyang Technological University

Dectin-1 is a receptor on conventional dendritic cells that is involved in the innate immune system recognition and processing of fungi and mycobacterium. Its activation induces kinases Syk and subsequently PLCγ, which can increase Ca2+ level in the cells. Advances in technology have enabled the detection and quantification of Ca2+ upon receptor activation through the use of flow cytometry techniques. In this project, a protocol for Ca2+ detection was established using Ca2+ indicator Indo-1. The process of protocol set-up involved troubleshooting works with sample mixing, temperature and gating flow. Subsequent application of the protocol illustrated transient Ca2+ fluxes inductions in Flt-3L BMDCs (bone marrow-derived dendritic cells) upon stimulation by TLR agonists LPS, Pam3Csk and Zymosan. However, Curdlan (pure Dectin-1 agonist) –induced Ca2+ flux was diminutive in BMDCs. As such, Dectin-1 overexpressing Nup98-HOXB4 Flt-3L derived-DCs were utilised to investigate Dectin-1 mediated Ca2+ flux in DCs. This was indeed the case as Curdlan was clearly observed to induce sustained Ca2+ flux, which provided direct evidence of Dectin-1-induced Ca2+ flux in the cells. However, the percentage of cells responding to TLR/Dectin-1 -agonists and GM-CSF combination by Ca2+ flux were diminished by 5-10%. This raised questions of GM-CSF role in receptor-induced signalling pathways.