Nanoscience and Microsystems ETDs

Author

Mario Paz

Publication Date

6-26-2015

Abstract

Understanding cellular signaling is important to learning about life at a fundamental level. As an extension of this, understanding the dysregulation of cell signaling is the key to identifying and treating disease. At present, there are multitudes of ways to investigate the cell signaling process, from immunofluorescence to single particle tracking. These techniques are advantageous in their own right, but having the ability to interrogate suspension and adherent cells with reagents at high temporal resolution and image them simultaneously is a process that could uncover many unseen signaling events. In Aim 1, we describe a microfluidic device for live cell imaging of cell signaling events after ligand activation. This investigation method is able to capture early events with a high temporal resolution. When coupled with fluorescence microscopy the responses of single cells loaded in the device can be measured using fluorescent reporters. In Aim 2, we have measured with high temporal resolution the calcium response of mast cells exposed to stimulus. Cells labeled with the calcium indicator dye, Fluo-4, are loaded into the microfluidic traps. By monitoring the intensity of emitted fluorescence from the excited Fluo-4, the amount of intracellular calcium can be measured. When exposed to the stimulus, we observe a change in the fluorescence emission, indicating calcium flux upon ligand activation. This study focuses on the use of a microfluidic device supplied by Sandia National Laboratoriess to track the cytoplasmic calcium changes due to ligand addition for the Fc\u0511R1 pathway in Rat Basophilic Leukemia (RBL) cells. Specifically the investigation will look at stimulation pulses over a range of duration, and compare single stimulation pulses to multiple pulses in order to achieve a more in depth understanding this signaling pathway.

Keywords

cell signaling, microfluidics, calcium signaling, allergic response, fluorescence microscopy

Sponsors

National Institutes of Health, National Science Foundation

Document Type

Thesis

Language

English

Degree Name

Nanoscience and Microsystems

Level of Degree

Masters

Department Name

Nanoscience and Microsystems

First Advisor

Lidke, Diane

First Committee Member (Chair)

James, Conrad

Second Committee Member

Chi, Eva

Third Committee Member

Evans, Deborah

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