Biology Reference
In-Depth Information
3. The Use of
Fluo-4 to Measure
Ca
2+
Sparks in
Cerebral Artery
Myocytes
Single-wavelength Ca
2+
indicators, such as fl uo-4, or its analogue
fl uo-3, have become widely used for imaging of fast, small volume
Ca
2+
signaling events, such as Ca
2+
sparks (
23
). One advantage of
fl uo-4 over ratiometric indicators (e.g., fura-2) in the measurement
subcellular Ca
2+
signaling events is superior signal-to-noise ratio.
Fluo-4 is excited at 488 nm and exhibits increased fl uorescence
intensity (>100-fold) upon binding Ca
2+
without a shift in absorp-
tion or emission spectra (
24
). Moreover, fl uo-4 has a lower affi nity
for Ca
2+
than fura-2 (
K
d
= 400 nM compared with
K
d
= 282 nM for
fura-2) and fast binding and unbinding kinetics, making it suitable
for tracking rapid Ca
2+
signaling events (
24
). Ca
2+
sparks can be
measured using a number of imaging approaches, including single
photon and multiphoton confocal microscopy, total internal refl ec-
tion fl uorescence (TIRF) microscopy, and wide-fi eld microscopy
using low-noise CCD cameras (
23
).
Previously, the temporal resolution of laser-scanning confocal
microscope systems suffi cient to measure Ca
2+
sparks could only be
achieved using line-scan mode, as opposed to two-dimensional
(2D)
x
-
y
image scanning (
5
). However, the interpretation of
images obtained using line-scanning is complicated by uncertain-
ties in the position of the scan line with regard to the center of the
Ca
2+
release event resulting in a loss of spatial information, such as
accurate determination of Ca
2+
spark area. Another disadvantage of
this method is the inability to measure the total number of active
spark sites within a cell. Currently, a number of laser-scanning con-
focal microscope systems with suffi cient 2D sampling frequency
(>30 Hz) are available. The following section provides a detailed
description of protocols that we have used to measure Ca
2+
sparks
in cerebral artery myocytes using fl uo-4 and 2D laser-scanning
confocal microscopy.
3.1. Background
Cell chamber with glass coverslip bottom
Cell-permeant fl uo-4AM (Invitrogen)
3.2. Materials
and Instruments
●
●
Laser scanning confocal system (e.g., Oz, Noran Instruments)
●
Inverted fl uorescence microscope (Nikon Diaphot)
●
60×/1.2 NA water-immersion objective lens
●
Krypton/argon laser (480 nm is used to excite fl uo-4)
●
Dark room
●
3.3. Procedures
Cerebral arteries are enzymatically dissociated to obtain individual
smooth muscle cells (Fig.
3
) (
25
). After dissection, cerebral arter-
ies are incubated in a glutamate-containing isolation solution
(GIS) of the following composition (in mM): 55 NaCl, 5.6 KCl,
3.3.1. Isolation of Cerebral
artery Myocytes
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