WPI MPS-4, Инструкция по эксплуатации, Страница: 16 / 36

12 World Precision Instruments
1 to 8 in sequence, until the water droplets of each channel comes out of the
micro perfusion head continuously.
6. Determine the flow velocity at the micro perfusion head from each channel
using a stopwatch. Flow velocity for different channels should be about the
same. Otherwise, check for air leakage or residual bubbles in the corresponding
channel.
Testing Drug Delivery
The drug perfusion area of the MPS-4 series can cover the whole view field of
a 200X microscope (objective 20X, eyepiece 10X). However, in order to perform
the experiments in an effective and reliable way, we suggest several preliminary
experiments as a control result. The following procedure uses patch clamp as an
example.
1. Clean the tubing system with distilled water. “Cleaning” on page 13.
2. Fill channel 1 with 150mM filtered NaCl solution. Fill the other channels with
distilled water. Check the system (bubble and flow velocity) as previously
described in “Hardware Testing Procedure” on page 11.
3. Fill the culture dish with NaCl solution, and place it on the microscope stage
4. Adjust the position of the perfusion head using the micromanipulator, so that
the tip of the micro perfusion head is close to the bottom of the dish. The access
angle is about 35-45°.
5. Pull a 1µm micropipette. Fill the pipette with 150mM NaCl solution to make it
a microelectrode, and connect it to a patch clamp amplifier. Use the electrode
micromanipulator to position the tip of the electrode right in front of the
perfusion head at the bottom of the glass dish.
6. Apply a 5–10mV voltage between the microelectrode and reference electrode,
and an electric current can be observed passing the electrode. Turning on any
of the distilled water filled channels should cause a rapid decrease of electric
current to zero. Turning off the distilled water and turning on channel 1 brings the
electric current back up to its original level. Test the rest of the channels and the
results should be the same.
7. If the electrode current does not reduce to zero, adjust the position and direction
of the perfusion head and electrode, and repeat step 6. After several tests, you
will get an idea about the right position and direction of the perfusion head, cell
and electrode.
8. After the test, clean the entire tubing system.
9. For a formal experiment, the drug perfusion procedures are almost the same as
above, except that the NaCl solution is replaced by drug solutions, and only the
optimal positions for perfusion head, cell and electrodes are used.