Running The Pampa Assay - Beckman Coulter Biomek FX-ADMETox Manual Del Usuario

Quick Start Guide
Using the PAMPA EVOLUTION96 Software and Biomek FX Workstation

Running the 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.
.pHstart =2.6
pHtarget
5.0
6.2
7.4
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:
1 2
pH pH
7.4 7.4
2. Prepare GIT-Lipid.
a. Thaw 2 glass ampules of GIT lipid solution.
b. Break ampules 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.
4. Prepare test compounds
a. Spectroscopic purity DMSO for test compound preparation.
b. Add DMSO to test compounds according to the table below:
6
.pHstart=2.8
NaOH (mL) pHtarget
5.5 5.0
8.3 6.2
11 7.4
3 4 5 6
pH pH pH pH
7.4 7.4 6.2 6.2
.
.
.
.
NaOH .pHstart=3.0
(mL) pHtarget
5 5.0
7.8 6.2
10.5 7.4
7 8 9 10
pH pH pH pH
6.2 6.2 5.0 5.0
Biomek FX/PAMPA Evolution96 Quick Start and Trouble Shooting Guides
NaOH (mL)
4.5
7.3
10.2
11 12
pH pH
5.0 5.0
PN A29703AB