F
LEXCELL
®
I
NTERNATIONAL
C
ORPORATION
20
tubing size and pump assigned to it. Modify
shear stresses if necessary.
4.
The regime is now ready to run.
S
ETTING UP AN
E
XPERIMENT
1.
Set up the system in an incubator according
to instructions on page 2.
2.
Sterilize the Streamer
®
unit according to
instructions on page 2. Close the lid and
tighten screws until the lid is flush with the
body of the device.
3.
Place the Streamer
®
in the incubator with
the remainder of the system. This will keep
the temperature of the unit at 37
°
C.
4.
Culture cells on 6 Culture Slips
®
.
Be sure
that you culture on the side with the brown
Teflon
®
rim printed around the borders.
Be careful to plate cells only within this rim.
Allow cells at least 48 hours for full
attachment to slides.
5.
Create your regime in the StreamSoft
software.
After cells have attached to slides:
1.
Put one bottle of PBS into the system
medium container.
2.
Pump the PBS through the system to flush
the tubing and Streamer
®
device, then
discard the perfusate; this is done to remove
any cytotoxic substances that may have
accumulated during sterilization.
3.
Put 500 ml of medium into the medium
container (this may be adjusted later as you
determine your system volume
requirements).
4.
Flow the medium through the system to
flush out remaining PBS. Remove medium
and replace with 500 ml of fresh sterile
tissue culture medium.
5.
Pump medium through the entire system to
fill the flow device and tubing. 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.
6.
As the flow continues, check to be sure that
no air bubbles are 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.
7.
Once the tubing and flow device are filled
with medium and all air bubbles are
eliminated, stop flow, then reverse flow so
that the medium is drawn down to about
80% of the Streamer
®
body. The fluid level
will have to be estimated once the fluid
flows past the Streamer
®
outlet fitting.
When the fluid reaches this level, stop the
flow again.
8.
Tighten the 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.
9.
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.
10.
Remove the Streamer
®
screws and open the
hinged top.
11.
Transfer your cells from the incubator to the
tissue culture hood.
12.
Using forceps and/or your fingers with
sterile gloves, grasp a Culture Slip
®
at one
