F
LEXCELL
®
I
NTERNATIONAL
C
ORPORATION
4
Regime
and
Hardware
, then click
Update
.
The regimen is now ready to start.
7.
Culture cells on six Culture Slips
®
. Be sure
that you culture on the side with the Teflon
®
rim printed around the borders. Be careful
to plate cells only within this rim. We
recommend allowing cells at least 48 hours
to attach to slides before beginning your
flow experiment.
After cells have attached to slides:
8.
Be sure that the Streamer
®
is closed (the top
lid should be flush with the body of the
device).
9.
Place 500 ml of PBS into the medium
container and pump through the system to
flush out impurities. This can be done by
starting your regime or using the
manual
mode
under the
Operate
menu in the
software. If you are not using the software,
set the pump to the appropriate tubing size,
set the flowrate at 300 ml/min and press the
start button.
10.
After pumping for several minutes, remove
the PBS from the medium container and
replace with 500 ml of sterile tissue culture
medium.
11.
Pump the culture medium through the
system to flush out remaining PBS.
Remove the medium and replace with 500
ml of fresh sterile tissue culture medium.
12.
Pump the tissue culture medium through
the entire system. Once the system is full,
tilt the pulse dampeners, one at a time, at an
angle of approximately 20 degrees, such
that the direction of the flow is going from
the vertex of the angle to the open end of the
angle. Leave the pulse dampener in this
position until the fluid comes through the
outlet fitting again, then lay the pulse
dampener down horizontally. This process
will allow the pulse dampener to fill to a
level slightly higher than the fittings,
thereby creating a bubble trap for any air
bubbles that may accidentally enter the
system. Do the same with the second pulse
dampener. Once this process is complete,
allow flow to continue and go to the next
step.
13.
As the flow continues, check for any air
bubbles visibly trapped within the tubing.
Also check the walls of the medium
container to be sure that no air bubbles have
formed on the sides. If so, swirl the medium
around to release air bubbles from the side
walls.
14.
Once the tubing and flow device are filled
with medium and all air bubbles are
eliminated, reverse the flow direction on the
pump to draw the medium level back to the
flow chamber and down past the head of the
chamber, then stop the pump. The fluid
level will have to be estimated once the
fluid can no longer be seen in the tubing
coming from the head of the Streamer
®
.
15.
Tighten the small clamp on the Phar-Med
®
tubing just to the right of the pump head so
that the flow path in the tubing is
completely closed off.
16.
Turn the lever arm on the MasterFlex
pump all the way to the left to release the
tubing and remove the tubing from the
pump head. Carefully move the tray
containing the Streamer
®
device, tubing,
pulse dampeners, and fluid collection
reservoir to the tissue culture hood.
17.
Remove the Streamer
®
screws and open the
hinged top.
18.
Transfer your cells from the incubator to the
tissue culture hood.
19.
Using forceps and/or your fingers with
sterile gloves, pick up each Culture Slip
®
and place it into each one of the slots in the
flow device.
Be sure that the side of the
slide with cells attached is facing the flow
area adjacent to the slot, not the closed
wall of the slot
. Gently slide each Culture
Slip
®
downward until it reaches the bottom
of its chamber. Be careful that the Culture
Slip
®
glass is not chipped against the
stainless steel surface during this process.
