Biomedical Engineering Reference
In-Depth Information
Fig. 1
Visualization of fluorescence recovery in cell-gel constructs before photobleaching (
t
ini
),
after photobleaching (
t
0
), 20 minutes after photobleaching (
t
20
), and 30 minutes after photobleach-
ing (
t
30
).
Red circle
indicates photobleached cell,
white arrow
indicates reference cell
of cells are irreversibly photobleached, causing a concentration gradient to form. If
the bleached cell communicates with adjacent cells by means of gap junctions, this
concentration gradient will be equilibrated. This equilibration is detected by moni-
toring fluorescence recovery (Fig.
1
). It has been shown that calcein-AM is a good
molecule for discriminating fluorescence recovery in short duration experiments. IF
is a technique that enables the visualization of a specific protein or antigen in cells
by binding a specific antibody that is fluorescently labeled.
9.1 Methods
Murine embryonic stem cells were maintained in T75 culture flasks, and after 3 to
4 passages, 1 million cells were spun down and resuspended in media containing
pro-osteoblastic beta-glycerol phosphate (BGP) (260 mg/ml of media). The cells
were then combined with purified bovine collagen I (Advanced BioMatrix, 800 µl
collagen/1 ml cell-gel construct), seeded in 24-well plates, and incubated at 37 °C
for 5, 15, 20, and 30 days. This protocol has been previously shown by our group to
be sufficient for embryonic stem cell differentiation into osteoblasts in collagen gel
constructs [
65
].
After 5, 15, 20, and 30 days of differentiation, cell-gel constructs were incubated
at 37 °C with 500 µM calcein AM, or 1 mM octanol (a non-specific blocker of inter-
cellular communication) for two hours. Constructs were also incubated in 100 µM
α
-glycyrrhetinic acid (a specific gap junction inhibitor) for 45 minutes to determine
what effect specific gap junction blocking had on the ability of cells to communicate.
A Zeiss LSM510 microscope was used for imaging. An initial image was recorded
(
t
ini
) at 20 % laser intensity. An oval region of interest (ROI) was then fit around
the cell body and any visible processes of a cell. This ROI was then photobleached
at 100 % laser intensity for 10 to 15 seconds. Images were obtained immediately
after photobleaching (
t
0
), after 2 min (
t
2
), 5 min (
t
5
), 10 min (
t
10
), 20 min (
t
20
),
and 30 min (
t
30
) of recovery. The mean pixel intensity within cells was determined
using NIH ImageJ 1.43 software and normalized to an uninvolved cell in the image
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