Biology Reference
In-Depth Information
2. The Use of
Fura-2 to Measure
Global [Ca
2+
]
i
in Cerebral artery
Smooth Muscle
The most popular technique for quantifying intracellular-free Ca
2+
is by monitoring the fl uorescence of Ca
2+
-sensitive indicator dyes
(
16
). The dual-wavelength ratiometric indicator fura-2 is currently
the most widely used indicator for the measurement of global
[Ca
2+
]
i
in isolated cerebral artery myocytes and intact pressurized
vessels. Fura-2 shows 1:1 Ca
2+
binding stoichiometry and under-
goes a signifi cant shift in excitation spectra depending on whether
or not Ca
2+
is bound (
l
bound
= 340 nm;
l
free
= 380 nm) while emis-
sion spectra (510 nm) is independent of Ca
2+
binding to the dye (
17
).
Changes in fl uorescence ratios are therefore independent of inten-
sity, eliminating potentially confounding factors, such as differen-
tial dye loading, photobleaching, or time-dependent expulsion of
the dye by the cell. As a result, fura-2 is preferred over single-
wavelength intensity-modulating indicators for quantifi cation of
absolute intracellular Ca
2+
concentrations. In this section, we
describe techniques used for measuring global [Ca
2+
]
i
in intact
pressurized cerebral arteries using fura-2.
2.1. Background
5 ml myograph chamber (Fig.
1a
) (Living Systems Instru-
mentation)
Fine-tipped forceps (Fine Science Tools)
2.2. Materials
and Instruments
●
●
Cell-permeant fura-2AM (Invitrogen)
●
Calcium and diameter recording system (e.g., IonOptix Inc.):
●
-
Hyperswitch light source
-
Xenon arc lamp
-
IonWizard-Core and Analysis Soft Edge software
-
Fluorescence system interface
-
Myocam-S digital CCD video camera
-
Photomultiplier tube (PMT) subsystem
-
Dichroic mirror
-
Inverted fl uorescence microscope
Photon-to-voltage converter
●
20×/0.50 NA objective lens
●
Peristaltic pump (Ismatec Inc.)
●
PS-200 pressure servo controller with peristaltic pump (Living
●
Systems Instrumentation)
Dark room/red light
●
Cerebral arteries and arterioles (50-200
m in diameter) are dis-
sected from the brain and kept in ice-cold artifi cial cerebral spinal
fl uid (aCSF) of the following composition (in mM): 125 NaCl,
μ
2.3. Procedures
2.3.1. Tissue Preparation
and Dye Loading
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