Biology Reference
In-Depth Information
CHAPTER 5
Practical Aspects of Measuring Intracellular
Calcium Signals with Fluorescent Indicators
Joseph P. Y. Kao, Gong Li, and Darryl A. Auston
Center for Biomedical Engineering and Technology, and
Department of Physiology
University of Maryland School of Medicine
Baltimore, Maryland, USA
Abstract
I. Introduction
II. Fluorescent Ca
2
þ
Indicators
III. Loading Indicators into Cells
A. Limited Aqueous Solubility of AM Esters
B. Dye Compartmentalization: Loading of Indicator into Subcellular
Compartments Other than the Cytosol
C. Dye Leakage or Extrusion from Cells
D. Procedure for Loading
IV. Manipulation of [Ca
2
þ
]
A. Using EGTA and BAPTA as Extracellular Ca
2
þ
Bu
V
ers
B. Lowering Extracellular [Ca
2
þ
]
C. Divalent Cation Ionophores
D. Bu
V
ering Changes in Intracellular [Ca
2
þ
]
V. Conversion of Indicator Fluorescence Signal into Values of [Ca
2
þ
]
A. Calibrating a Nonratiometric Fluorescent Indicator
B. Calibrating a Ratiometric Fluorescent Indicator
VI. Reporting Indicator Fluorescence Intensity Changes without Calibration
A. Reporting Relative Changes in Fluorescence:
F
/
F
0
and
D
F
/
F
0
B. Caveat in Interpreting Relative Fluorescence Changes: Indicator
Fluorescence is Not a Linear Function of [Ca
2
þ
]
VII. Measuring [Ca
2
þ
] in Mitochondria
A. Estimating the Fraction of Intracellular Rhod-2 Indicator that Resides in
Mitochondria
B. Minimizing Rhod-2 Loading in the Cytosol
C. Monitoring Cytosolic and Mitochondrial [Ca
2
þ
] Simultaneously
