eBook: Advanced Tools Transforming Neurology Research - 20

intercellular G protein-coupled receptors to release
intracellular calcium ions.8
Application
Cell preparation
The application used combined bovine aortic endothelial
cells (BAEC) with Fluo-8® AM, which is a
cell-permeable calcium indicator assay. BAEC is a
reputable endothelial cell line that efficiently provides
a model system to measure calcium permeability
of the cellular characteristics. To evaluate
the calcium ion efflux, the healthy BAEC were first
seeded in the media of DMEM with 10 % fetal bovine
serum and penicillin-streptomycin overnight
at 20,000 cells/100 μl/well in a 96-well plate (SPL,
30296) for 16 hours. The culture media were aspirated,
and the cells were incubated with 5 μM of
Fluo-8® AM and 2.5 mM ReadiUse™ probenecid in
Hanks and Hepes buffer (HHBS) at room temperature
(protected from light) for one-hour incubation
(AATbio.com Cat. 21080 & Cat. 20062). Then, the
media was removed, and the wells were washed
with 100 µL DPBS according to the manufacturer's
instructions. The 500 μM stock solution of ATP
in DPBS was prepared in advance, and the 20 μl of
ATP stock was ready to add on each well of 96-well
plate to make ATP final concentration of 20 μM
(Fishersci, Cat AC1028001000). For acquisition of
images, the cell plate was placed on the stage of
the CELENA® X system and set CELENA® X Explorer
time-lapse function for 3-min scanning of images
and capturing speed of 4 images in a second (4 fps,
as fast as possible) with 10X objective. The scanning
started immediately after gently applying the
20μl of the prepared ATP stock to each well of the
plate. Next, the analysis was performed through the
pipeline with the six following modules to quantify
membrane potential of calcium mobilization;
1. AddsingleImage module to select an image(Time
0) that will be used to create a mask image,2.
IdentifyprimaryObjects module to find the individual
cells in Time 0 image by the boundary of fluorescent
signals. Next 3. MaskImage module to mask
image using the object identified in the previous
step, 4. MeasureObjectIntensity module to measure
and quantify the intensity of the fluorescent,
5. GrayToColor module to convert mono images to
green pseudocolor images, and 6. OverlayOutlines
module to draw the outlines each cell. They are using
the result file saved as .CSV format, the calcium
mobilization of the total and several represented
cells were plotted over time (Figure 1). The selected
time-lapse images of intracellular calcium changes
were presented at 0 s, 10 s, 20 s, 40 s, and 160 s,
and they were exhibited the Fluo-8® signals elicited
higher levels of transient during 10 to 40 second
in intracellular calcium ion responding fluorescent,
and the highest total intensity was plotted on the
35 s (Figure F).
Conclusion
In the presence of ATP, intracellular calcium ion
was instantly augmented and then gradually returned
to baseline levels over 2-3 minutes from
the intensity of the total fluorescent. In the color
coordinated three cases, the individual cells may
not correctly synchronize their intensity on the 35s
but scuttled between 15s to 45s. Thus, the results
based on the measurement of the intensity over 3
minutes represented the calcium values of changes
over time and peak heights in 10-40 second
within 180 seconds.9
Since the reaction occurs in
such a short time, delivering the agonist stimulator
20
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