© 2005 Beckman Coulter, Inc.
4300 N. Harbor Blvd., Fullerton, CA 92834-3100
Printed in U.S.A.
Biomek FX-ADMETox
Workstation
PAMPA Evolution96
Permeability Analyzer
Quick Start Guide
A28712-AA
July 2005
Quick Start Preliminaries for Using the
PAMPA EVOLUTION96 Software and
Biomek FX Workstation
Preparing a Blank
Default file which contains the UV spectra of a Blank may be
replaced with new one.
1.
Launch PAMPA Evolution-96 Command Software on the
computer connected to the PAMPA Evolution
instrument.
2. Go to menu
File | Save as
and save new file as
""YYMMDD_Blank" (for example: 040701_Blank) where
the first 6 characters are the year-month-day numbers.
3. Be careful not to touch the underside of the UV plates,
place 4 new 96-well plastic UV plates stacked on top of
each other into position P 11.
4. Put High Profile, 12 Trough Reservoir filled with System
Solution into position P4.
5. Click the
Start Assay
button on the toolbar. The Start
Assay dialog displays. Make sure that all check-boxes
are not selected.
6. Click the
Start
button.
7. A dialog box displays
Transfer the System Solution to
UV plate.
When the instrument finishes this step, Spectrophotometer
will start reading UV spectra of the blank plate. Wait till
Spectrophotometer finishes reading.
1.
To inspect the spectrophotometric data, click
View 96
Spectra
button.
2. 96 spectra view displays.
3. Inspect UV spectra of the blank in order to exclude
bacterial contamination.
4. In 96 spectra view go to menu
File | Export Plate
Spectra
5. The
Save Spectra
dialog box displays.
6. Select
Blank Plate
from
Plate to be Exported
drop-
down list.
7. Click the
Save
button.
8. A special spectra file will be saved in the same folder
where data file is under the name
Data file name_Blank
Plate
. If appropriate care is taken of the System
Solution, one Blank may be reused for many
experiments.
9. Abort the assay and close the method.
10. Start a new PAMPA method and go to the main menu
Instrument | Settings
.
11. The internal UV blank spectrum must be loaded into the
file.
12. Browse for the file
Data file name_Blank Plate.spc
and
click the
Open
button.
13. Select
Use pre-measured blank
.
14. Select
Tell me about low -UV wells...
15. Select
Auto-refine
. The results of the experiment will
have the results automatically calculated.
16. Click the
Ok
button when done.
Run PAMPA Assay
1.
Prepare system buffer with different pH conditions
.
a.Prepare 1 Liter of System Solution by adding 25 mL of
System Solution Concentrate to total 1 L of distilled
water using a graduated cylinder
.
b.Pour equal aliquots of System Solution in 3 clean
glass bottles (about 333 mL each)
c.Measure initial pH of System Solution using pH meter
.
d.Adjust pH to 7.4, 6.2 and 5.0 by adding 0.5M NaOH
according to the table below.
e.Verify System Solution pH using pH meter.
f.Add 15 mL of the pH-adjusted System Solution in each
partition of the High Profile 12 Trough Reservoir
according to the table below
:
2. Prepare GIT-Lipid.
a.Thaw 2 glass ampules of GIT lipid solution.
b.Break amples using ampule breaker.
c.Empty GIT lipid into the first column of a Low Profile
12 Trough Reservoir.
d.Transfer 18 µL of lipid into each well of the LipidPlate.
3. Prepare Acceptor System Buffer (ASB 7.4).
a.Place 50 mL of ASB 7.4 into a Low Profile Reservoir
.
TM
.pHstart
=2.6
.pHstart=
2.8
.pHstart=3.0
pHtarget
NaOH
(mL)
pHtarget
NaOH
(mL)
pHtarget
NaOH
(mL)
5.0
5.5
5.0
5
5.0
4.5
6.2
8.3
6.2
7.8
6.2
7.3
7.4
11
7.4
10.5
7.4
10.2
1
2
3
4
5
6
7
8
9
10
11
12
pH
7.4
pH
7.4
pH
7.4
pH
7.4
pH
6.2
pH
6.2
pH
6.2
pH
6.2
pH
5.0
pH
5.0
pH
5.0
pH
5.0
