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TECHNICAL BRIEFS

# A Novel Single-Photon Counting Technique Applied to Highly Sensitive Measurement of $[Ca2+]i$ Transient in Human Aortic Smooth Muscle Cells

[+] Author and Article Information

Energy Phenomena Laboratory, Department of Mechanical Engineering and Science, Tokyo Institute of Technology, 2-12-1 Meguro-ku, Tokyo 152-8859, Japanstada@mech.titech.ac.jp

Ken Okazaki

Energy Phenomena Laboratory, Department of Mechanical Engineering and Science, Tokyo Institute of Technology, 2-12-1 Meguro-ku, Tokyo 152-8859, Japan

J Biomech Eng 128(5), 777-781 (Apr 28, 2006) (5 pages) doi:10.1115/1.2264396 History: Received March 16, 2005; Revised April 28, 2006

## Abstract

This study demonstrates that aequorin, a luminescent natural dye, is useful for vascular cell intracellular $Ca2+$ concentration $([Ca2+]i)$ determination. A new single-photon counting technique was developed to resolve the effects of fluid flow shear stress on $[Ca2+]i$ in human aortic smooth muscle cells (HASMCs). Confluent HASMCs were grown on petri dishes loaded with aequorin. Then the dishes were placed in a luminometer chamber after the physiological level of shear stress was applied to the HASMC surfaces. The chamber was housed inside a highly sensitive photomultiplier tube. It detected ultraweak photon emission in response to the $[Ca2+]i$ transient. In the presence of $2.0mM$ extracellular $Ca2+$, a shear stress of $12dyn∕cm2$, applied for $60s$ to the top surface of the HASMC monolayer, elicited a sharp increase in $[Ca2+]i$.

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## Figures

Figure 1

(a) Light-shield housing. (b) Diagram of shear loading apparatus for exposing HASMCs to shear stress on a collagen-coated 35-mm petri dish. (c) A block diagram of the highly sensitive photon-counting system.

Figure 3

(Top) Effect of shear stress of 16dyn∕cm2 on [Ca2+]i. (Bottom) Time variation of light emission intensity of [Ca2+]i in the absence of fluid flow shear stress. Graphs are the average light emission response of four experiments. Error bars represent mean ±SD. ∗, significant difference (P<0.01) from the Control group.

Figure 2

Transient of the intensity of biophoton. “HASMCs” in the figure denotes the intensity of biophoton emission from HASMCs whereas “Control” denotes that of background noises.

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