Cytochrome P450 Assay on the
Agilent BioCel 900 System
Application Bulletin
The Agilent BioCel 900 Automation Platform
P-450 substrate
(non-fluorescent)
R1 R2 +R1 +R2
P-450 substrate
(fluorescent)
P-450 CYPXXX
Overview of the metabolism of the fluogenic “blocked” dye into a Fluorescent Metabolite (as outlined in:
Invitrogen: Lit #Lo5o4 Rev. 06003)
Summary
• Small footprint automation system pro-
vides capacity for 20 microplates, without
manual intervention
• Microplate processing time about x hour
for a 20 plate run
• Efficient scheduling maximizes throughput
and ensures same treatment for
each plate
Introduction
Cytochrome P450 (CYP450) enzymes are
the largest group of drug-metabolizing
enzymes and therefore critical targets
for drug discovery. Inhibiting these
enzymes is an important part of
ADME-Tox compound profiling. The
demand for this type of assay is high
because of the increasing output from
high-throughput screening (HTS) and the
pressure to frontload as much testing as
possible during drug discovery. There
are a number of different approaches
for CYP450 testing. In this assay, the
CYP450 enzyme metabolizes a fluoro-
genic substrate (step 1) into a highly
fluorescent product in the presence of
an NADPH regenerating system. The
fluorescent signal (step 2) is directly
proportional to the enzyme activity.
Addition of CYP450 inhibitors results in
a reduction in enzyme activity.
3-Axis Robot
Agilent Microplate Centrifuge
Plate Hotels
Agilent PlateLoc Sealer
Agilent Labware Stackers
Agilent Bravo Platform
System Description
The Invitrogen Vivid kit is easily adapted for
automation on the Agilent BioCel System.
All liquid handling steps are carried out by
the Agilent Bravo Automated Liquid
Handling Platform. Pipetting can be per-
formed without tip touching, preventing
cross-contamination. Efficient mixing is gen-
erated by the Orbital Shaking Station, directly
after reagent pipetting. The Plate Hotels
store the assay plates during the run and for
the room-temperature incubation step.
Sample plates reside in Agilent Labware
Stackers before and after processing.
Agilent VWorks Automation Control software
manages all processes and incubation
times, to ensure reliable and repeatable
results. This application bulletin outlines a
protocol for the Invitrogen Vivid CYP450
Screening kit using the BioCel 900 System.
Materials
Component List
• Bravo Platform with 384ST disposable-tip
head, 3 Reservoirs, 1 Tip Wash Station,
1 Orbital Shaking Station
• Two Labware Stackers
• Two Plate Hotels
• Agilent PlateLoc Thermal
Microplate Sealer
• Agilent Microplate Centrifuge
• Tecan Spectrafluor plus
Labware List
• Microplate A (Assay Plate):
Greiner 384PS, black, flat-bottom
• Microplate B (Sample Plate):
Greiner 384PP, v-bottom
• Tipbox A, B, C, D: Agilent Tips 384 ST 70 µL
Reagent List
• Reservoir A: Cyp P450/NADPH
regenerating mix
• Reservoir B: Substrate/NADP+ mix
• Reservoir C: Stop reagent
www.agilent.com/lifesciences/automation
This item is intended for Research Use Only.
Not for use in diagnostic procedures. Information,
descriptions, and specifications in this publication
are subject to change without notice.
Agilent Technologies shall not be liable for errors
contained herein or for incidental or consequential
damages in connection with the furnishing,
performance, or use of this material.
Invitrogen and Vivid are registered trademarks of
Invitrogen Corporation.
© Agilent Technologies, Inc., 2009
Published in the U.S.A., February 26, 2009
Publication Number 5990-3551EN
Instrument Layout
The Agilent Bravo deck (top view).
One Orbital Shaking Station (location
9), four reservoirs (locations 1-3, 7),
and four tipboxes (locations 4-6, 8) are
placed manually on the deck before
the protocol is started.
Protocol Workflow
1. Move microplate B from Labware
Stacker 1 to Microplate Centrifuge.
2. Spin for 20 s.
3. Move microplate B from Microplate
Centrifuge to Bravo location 9.
4. Press on tips at location 8.
5. Aspirate 20 µL of sample from
microplate B.
6. Move microplate B from Bravo
location 9 to the PlateLoc.
7. Seal microplate B.
8. Move microplate B to Labware
Stacker 2.
9. Move microplate A from Plate
Hotel 1 to Bravo location 9.
10. Dispense 20 µL of sample into
microplate A.
11. Wash tips at location 7.
12. Release tips at location 8.
13. Press on tips at location 4.
14. Aspirate 20 µL Cyp450 mix from reser-
voir A and dispense into microplate A.
15. Shake 20 s.
16. Release tips at location 4.
17. Move microplate A from Bravo
location 9 to Plate Hotel 1.
18. Incubate for 20 min.
Conclusions
The Agilent BioCel 900 System provides a
robust, high-throughput solution for quan-
tifying Cytochrome P450 activity. The small
footprint and open-access of the BioCel
System makes it an ideal solution for medium
to high throughput ADME/Tox applications,
such as the CYP450 assay outlined here. All
microplates are handled and processed in the
same manner, including constant incubation
times, ensuring reliable and repeatable results.
19. Move microplate A from Plate
Hotel 1 to Bravo location 9.
20. Press on tips at location 5.
21. Aspirate 25 µL Substrate mix from
reservoir B and dispense into
microplate A.
22. Shake 20 s.
23. Release tips at location 5.
24. Move microplate A from Bravo
location 9 to the Spectrafluor device.
25. Read microplate A.
26. Move microplate A from the
Spectrafluor device to Plate Hotel 1.
27. Incubate 15 min.
28. Move microplate from Plate Hotel 1 to
Bravo location 9.
29. Press on tips at location 6.
30. Aspirate 25 µL Stop mix from
reservoir C and dispense into
microplate A.
31. Shake 20 s.
32. Release tips at location 6.
33. Move microplate A from location 9 to
the Spectrafluor device.
34. Read microplate A.
35. Move microplate A from the
Spectrafluor device to Plate Hotel 1.
Agilent Labware Stackers and Hotels (top view). Sample plates are stored in two Labware
Stackers (left), with a capacity of 60 plates per Labware Stacker. Assay plates are stored and
incubated in two Plate Hotels (right), with a capacity of 10 plates per Plate Hotel.