Beckman Coulter Biomek FX-ADMETox User manual

© 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 23456789101112
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

4. Prepare test compounds.
a.Spectroscopic purity DMSO for test compound
preparation.
b.Add DMSO to test compounds according to the table
below:
c.Add 80 µl of each test compound solution into each
well of the StockPlate.
d.Extra test compound can be frozen for future use.
5. Populate the worksurface with appropriate labwares.
a.Place 3 96-well UV plates on worksurface position.
b.Place Biomek P200 tip boxes on worksurface position.
c.Place PAMPA Sandwich Plate (pre-loaded) on
worksurface position.
d.Place SUPPORT Plate on worksurface position.
e.Place the StockPlate on worksurface position.
f.Place the LipidPlate on worksurface position.
g.Place ASB solution Low Profile Reservoir on
worksurface position.
h.Place System Buffer High Profile 12 Trough Reservoir
on worksurface position.
i.Place Deep well plate for diluted test compounds on
worksurface position.
6. Enter test compound information into Excel template file.
a.Launch Microsoft Excel application.
b.Select File | Open.
c.Browse to open PAMPA Template.xlt file located in
the PAMPA Evolution command Software directory.
d.Enter each compound in the same well location that it
was loaded in the StockPlate.
e.Physiochemical constants are optional.
f.Do not leave blank spaces in the Excel template sheet.
g.Rename the Excel file and save in DATA sub-folder in
the PAMPA Evolution command Software.
h.Exit the Excel application.
7. Run PAMPA assay.
a.Click the Import from Excel button on the toolbar.
b.The Input Compounds dialog box appears, click the
Browse button to import test compound Excel
template file.
c.Click the OK button for the Warning dialog box for
correct StockPlate type.
d.Verify the loaded compound names and
physiochemical constants, click the OK button to
continue.
e.Open the Instrument-Settings, select the Use pre-
measured blank.
f.Browse to select DefaultBlankPlate.spc.
g.Uncheck Tell me about low-UV wells…
h.Click the OK button and exit the Instrument Settings.
i.Click the General Information button, fill in the
appropriate experimental conditions, click the OK
button.
j.Click the Start Assay button.
k.Select the Uninterrupted Assay Flow and Use pre-
measured blank check boxes.
l.Verify that Double-Sink Protocol is selected, and
incubation time is set to “0.5 hr”.
m.Select Properties and verify the System Solution
Volume = 600 µL and Stirring be ABL = 40 µM.
n.Select Multi pH Assay and select pH Map.
o.Verify the updated layout of System Solution at
different pH.
p.Click the OK button and Start the assay to proceed.
Test Compounds MW Weight (mg) DMSO (ml) Concentration (mM)
Ketoprofen 254.3 2.54 1 10
Verapamil 491.1 4.91 1 10
Antipyrine 188.2 9.40 1 50
Metoprolol 684.8 6.85 1 10
Carbamazepine 236.3 2.36 1 10
Ranitidine 350.9 3.51 1 10
Propanolol 295.8 2.96 1 10
DMSO
Ketoprofen
Verapamil
Antipyrine
Metoprolol
Carbamazepine
Rantidine
Propranolol