Biology Reference
In-Depth Information
3.3.2. Dye Loading
Isolated myocytes are loaded with fl uo-4 by mixing equal volumes
(e.g., 250
l) of cell suspension and HEPES buffered physiological
saline solution (HEPES-PSS) of the following composition (in
mM): 134 NaCl, 6 KCl, 1 MgCl
2
, 1.8 CaCl
2
, 10 glucose, 10
HEPES (pH 7.4) containing fl uo-4 AM (20
μ
M), and pluronic
acid (0.072%). The cell suspension containing a fi nal concentration
of 10
μ
M fl uo-4 and 0.036% pluronic acid is placed in the record-
ing chamber to allow cells to attach to the glass coverslip on the
bottom of the chamber during the 30 min loading/deesterifi cation
period at room temperature. Cells are then washed three times
with HEPES-PSS.
μ
Initially, cells are located using bright fi eld illumination. Once a
myocyte has been centered in the scan fi eld, images of emitted
light (wavelengths >500 nm) are acquired at a frequency of 58 Hz
(every ~17 ms, 256 pixels × 256 pixels), and magnifi ed (×2) by
acquisition software (68
3.3.3. Ca
2+
Spark
Measurement
m for 256 pixels). Images are usually
acquired for a period of 10-20 s and saved for future analysis
(Fig.
4
). Laser exposure can lead to cell damage which typically
causes the cells to become much brighter in appearance and abol-
ish Ca
2+
spark activity. Excessive laser exposure can also lead to
photo-bleaching of the indicator dye. Thus, it is recommended to
combine the lowest laser intensity that provides good cell visualiza-
tion with relatively short (10-20 s) periods of laser exposure.
Measurement of Ca
2+
sparks can also be performed in intact
pressurized cerebral arteries (Fig.
5
) (
11, 14, 26
). After dissection,
intact arteries are loaded with fl uo-4AM (10
μ
M at room tempera-
ture for 1 h, with 0.036% pluronic acid) followed by brief washes
with aCSF (see Sect.
2.3
). Arteries are then cannulated, pressur-
ized (see Sect.
2.3
) and Ca
2+
sparks imaged using 2D laser-scanning
confocal microscopy as described above for isolated myocytes.
Measurement of Ca
2+
sparks in the intact tissue provides the ability
to simultaneously examine events in multiple cells under more
physiological conditions (i.e., at physiological intravascular pres-
sures). However, due to the presence of connective tissue and mul-
tiple cell layers, the signal-to-noise ratio is decreased in images
from intact tissue compared to isolated cells. Thus, imaging per-
formed on isolated cells is preferred for detailed analysis of the
spatio-temporal properties of Ca
2+
sparks.
μ
A variety of custom-written software packages provide the ability
to detect and analyze Ca
2+
sparks. Ca
2+
sparks are typically assessed
using 2.1
3.3.4. Analysis
of Ca
2+
Sparks
m analysis areas (centered over the Ca
2+
spark-
induced peak in fl uorescence intensity) and defi ned as fractional
fl uorescence changes (
F
/
F
0
) greater than 1.3 (
5, 25
). Background
fl uorescence intensity (
F
0
) is determined within analysis areas by
averaging the fi rst 30 consecutive images without Ca
2+
spark activ-
ity (e.g., see Fig.
4b
). Frequency is the most commonly used
μ
m × 2.1
μ
Search WWH ::
Custom Search