Vivid® CYP450 Screening Kits User Guide
Catalog no. P2856, P2857, P2858, P2859, P2860, P2861, P2862, P2863, P2864, P2969,
P2970, P2972, P3019, P3021, PV6140, PV6141, and PV6142
Shipping: Varies
Storage: Varies (see Kit Contents)
Protocol part no. O-13873-r1 (MAN0003095)
Rev. date: 24 April 2012

Table of Contents
Introduction ........................................................................................................................................... 1
Kit Contents........................................................................................................................................... 2
Materials Required but not Supplied ...................................................................................................................................... 4
Optional Materials not Supplied .............................................................................................................................................. 4

Storage and Stability ............................................................................................................................. 4
Assay Theory ......................................................................................................................................... 5
Vivid ® CYP450 High-Throughput Screening Assay Protocol .................................................................. 6
Thaw Reagents ............................................................................................................................................................................ 6
Reconstitute Vivid® Substrate and Fluorescent Standard ..................................................................................................... 7
Prepare 1X Vivid® CYP450 Reaction Buffer ............................................................................................................................ 8
Prepare Standard Curve (Optional) ......................................................................................................................................... 8
Prepare Test Compounds, Positive Inhibition Control, and Solvent Control .................................................................... 9
Dispense Test Compounds, Positive Inhibition Control, and Solvent Control ................................................................. 9
Prepare and Dispense Master Pre-Mix .................................................................................................................................... 9Pre-Incubate .............................................................................................................................................................................. 10
Start Reaction ............................................................................................................................................................................ 10
Measure Fluorescence .............................................................................................................................................................. 10

Suggested Protocol for the Analysis of Results .................................................................................. 11
Kinetic Assay Mode ................................................................................................................................................................. 11
Endpoint Assay Mode ............................................................................................................................................................. 11

Suggested Inhibitors ........................................................................................................................... 12
References .......................................................................................................................................... 12
Purchaser Notification ........................................................................................................................ 13

Introduction
Vivid® CYP450 Screening Kits enable rapid measurement of interactions between drug candidates and cytochrome
P450 enzymes using a simple “mix-and-read” fluorescent assay that is designed for high-throughput screening in
multiwell plates. These kits will allow investigators to rapidly identify compound-CYP450 interactions, eliminating
unsuitable compounds early in the drug discovery process. Vivid® CYP450 Screening Kits can also be used to generatepredictive structure-activity relationship models to guide medicinal chemists in their design of compounds.
Test compounds are analyzed by their capacity to inhibit the production of a fluorescent signal in reactions using
recombinant CYP450 isozymes and specific Vivid® Substrates. The availability of more than one structurally unrelated
fluorogenic Vivid® Substrate for CYP2C9, CYP2D6, CYP3A4, and CYP3A5 reduces the potential for false negatives orfalse positives that could result from substrate-dependent interactions.

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Kit Contents
Vivid® CYP450 Screening Kit

Contents
Vivid® CYP450 Reaction Buffer I

Vivid® CYP1A2 Blue
(Cat. no. P2863)®

CYP1A2 BACULOSOMES Plus Reagent

–80°C

P3024

0.1 mg–20°C, light protected

®

P2876

0.1 µmol–20°C, light protected

P2913

50 mL

RT

PV6137

0.5 nmol–80°C

Vivid CC Substrate

PV6143

0.5 mg–20°C, light protected

Vivid® Blue Fluorescent Standard

P2876

0.1 µmol–20°C, light protected

Vivid® CYP450 Reaction Buffer I

P2881

50 mL

RT

Vivid EOMCC Substrate®

CYP2A6 BACULOSOMES Plus Reagent®

®

CYP2B6 BACULOSOMES Plus Reagent

P3028

0.5 nmol-80 °C®

P2975

0.1 mg–20°C, light protected

®

P2876

0.1 µmol–20°C, light protected

P2913

50 mL

RT

Vivid BOMCC Substrate
Vivid Blue Fluorescent Standard®

Vivid CYP450 Reaction Buffer II®

Vivid CYP2C8 Green(Cat. no. PV6141)®

CYP2C8 BACULOSOMES Plus Reagent

PV61380.5 nmol–80°C

Vivid® DBOMF Substrate

P2974

0.1 mg–20°C, light protected

Vivid® Green Fluorescent Standard

P2875

0.1 µmol–20°C, light protected

P2913

50 mL

RT

®

Vivid CYP450 Reaction Buffer II
Vivid® CYP2C9 Blue
(Cat. no. P2861)®

CYP2C9 BACULOSOMES Plus Reagent

P2378

0.5 nmol

–80°C

®

P2975

0.1 mg–20°C, light protected

®

P2876

0.1 µmol–20°C, light protected®

Vivid CYP450 Reaction Buffer II

P2913

50 mL

RT

CYP2C9 BACULOSOMES® Plus Reagent

P2378

0.5 nmol–80°C

Vivid® BOMF Substrate

P2869

0.1 mg–20°C, light protected

P2875

0.1 µmol–20°C, light protected

P2913

50 mL

RT

Vivid BOMCC Substrate
Vivid Blue Fluorescent Standard®

Vivid CYP2C9 Green(Cat. no. P2860)®

Vivid Green Fluorescent Standard®

Vivid CYP450 Reaction Buffer II®

Vivid CYP2C9 Red(Cat. no. P2859)®

CYP2C9 BACULOSOMES Plus Reagent

P2378

0.5 nmol–80°C

Vivid® OOMR Substrate

P2868

0.1 mg–20°C, light protected

Vivid® Red Fluorescent Standard

P2874

0.1 µmol–20°C, light protected

P2913

50 mL

RT

P2570

0.5 nmol–80°C

Vivid EOMCC Substrate

P3024

0.1 mg–20°C, light protected

Vivid® Blue Fluorescent Standard

P2876

0.1 µmol–20°C, light protected

P2881

50 mL

RT

®

Vivid CYP450 Reaction Buffer II
Vivid® CYP2C19 Blue
(Cat. no. P2864)®

CYP2C19 BACULOSOMES Plus Reagent®

®

Vivid CYP450 Reaction Buffer I
Vivid® CYP2D6 Blue
(Cat. no. P2972)

RT

0.5 nmolVivid CYP450 Reaction Buffer II

Vivid® CYP2B6 Blue
(Cat. no. P3019)

Storage

50 mL

P2792

®

Vivid CYP2A6 Blue
(Cat. no. PV6140)

Quantity

P2881®

Vivid Blue Fluorescent Standard®

Part no.

®

CYP2D6 BACULOSOMES Plus Reagent

P2283

0.5 nmol

–80°C

®

P3024

0.1 mg–20°C, light protected

®

P2876

0.1 µmol–20°C, light protected

Vivid EOMCC Substrate
Vivid Blue Fluorescent Standard

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Vivid® CYP450 Screening Kits User Guide

Vivid® CYP450 Screening Kit

Contents

Part no.

Quantity

Storage

P2881

50 mL

RT

P2283

0.5 nmol–80°C

Vivid MOBFC Substrate

P2871

0.1 mg–20°C, light protected

Vivid® Cyan Fluorescent Standard

P2877

0.1 µmol–20°C, light protected

P2949

50 mL

RT

P2948

1.0 nmol

–80°C

P3024

0.1 mg–20°C, light protected®

Vivid CYP450 Reaction Buffer I®

Vivid CYP2D6 Cyan
(Cat. no. P2862)®

CYP2D6 BACULOSOMES Plus Reagent®

®

Vivid CYP450 Reaction Buffer III
Vivid® CYP2E1 Blue
(Cat. no. P3021)®

CYP2E1 BACULOSOMES Plus Reagent®

Vivid EOMCC Substrate®

Vivid® CYP2J2 Cyan
(Cat. no. PV6142)

Vivid Blue Fluorescent Standard

P2876

0.1 µmol–20°C, light protected

Vivid® CYP450 Reaction Buffer II

P2913

50 mL

RT

CYP2J2 BACULOSOMES® Plus Reagent

PV61390.5 nmol

–80°C

®

P2871

0.1 mg–20°C, light protected

®

P2877

0.1 µmol–20°C, light protected

P2881

50 mL

RT

P2377

0.5 nmol–80°C

Vivid BOMCC Substrate

P2975

0.1 mg–20°C, light protected

Vivid® Blue Fluorescent Standard

P2876

0.1 µmol–20°C, light protected

Vivid® CYP450 Reaction Buffer I

P2881

50 mL

RT

Vivid MOBFC Substrate
Vivid Cyan Fluorescent Standard®

Vivid CYP450 Reaction Buffer I
Vivid® CYP3A4 Blue
(Cat. no. P2858)

Vivid® CYP3A4 Green(Cat. no. P2857)®

CYP3A4 BACULOSOMES Plus Reagent®

®

CYP3A4 BACULOSOMES Plus Reagent

P2377

0.5 nmol

–80°C

®

P2974

0.1 mg–20°C, light protected

®

P2875

0.1 µmol–20°C, light protected

Vivid DBOMF Substrate
Vivid Green Fluorescent Standard®

®

Vivid CYP3A4 Red(Cat. no. P2856)

Vivid CYP450 Reaction Buffer I

P2881

50 mL

RT

CYP3A4 BACULOSOMES® Plus Reagent

P2377

0.5 nmol–80°C

Vivid® BOMR Substrate

P2865

0.1 mg–20°C, light protected

P2874

0.1 µmol–20°C, light protected

P2881

50 mL

RT

CYP3A5 BACULOSOMES Plus Reagent

P2512

0.5 nmol–80°C

Vivid® BOMCC Substrate

P2975

0.1 mg–20°C, light protected

Vivid® Blue Fluorescent Standard

P2876

0.1 µmol–20°C, light protected

P2881

50 mL

RT

®

Vivid Red Fluorescent Standard®

Vivid CYP450 Reaction Buffer I®

Vivid CYP3A5 Blue
(Cat. no. P2970)®

®

Vivid CYP450 Reaction Buffer I
Vivid® CYP3A5 Green(Cat. no. P2969)

Page 3 of 13®

CYP3A5 BACULOSOMES Plus Reagent

P2512

0.5 nmol

–80°C

®

P2974

0.1 mg–20°C, light protected

®

P2875

0.1 µmol–20°C, light protected

Vivid DBOMF Substrate
Vivid Green Fluorescent Standard

All kits also contain 0.5 mL Vivid® Regeneration System, 100X (Part no. P2878; 333 mM Glucose-6-phosphate and
30 U/mL Glucose-6-phosphate dehydrogenase in 100 mM potassium phosphate, pH 8.0) and 0.5 mL Vivid® NADP+(Part no. P2879; 10 mM NADP+ in 100 mM potassium phosphate, pH 8.0). Store both components at –80°C.
•

The Vivid® CYP450 Reaction Buffers are 200 mM (Reaction buffer I), 100 mM (Reaction buffer II), or
400 mM (Reaction buffer III) potassium phosphate pH 8.0.

•

CYP450 BACULOSOMES® Plus Reagents consist of recombinant human cytochrome P450, human
cytochrome P450 reductase, and in some cases, human cytochrome b5.

•

The Vivid® Substrates and Standards are supplied as a dried film. Reconstitution is necessary before use.

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Vivid® CYP450 Screening Kits User Guide

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Materials Required but not Supplied•

Multi-well black plates suitable for fluorescence measurements. We recommend using 96-well black,non-treated polystyrene assay plates (Corning/Costar, Cat. no. 3915).

•

Fluorescence plate reader with filters as described in Table 7 (page 11)•

Pipetting devices•

Reagent reservoirs•

Acetonitrile, anhydrous

•

DMSO

•

Nanopure water•

P450 inhibitor (see Suggested Inhibitors, page 12) for use as a control inhibitor

Optional Materials not Supplied•

0.5 M Tris base for use as a Stop Reagent. This solution can be prepared by dissolving 60.6 g of Tris base(e.g., Sigma-Aldrich, Cat. no. T1503) in 1 L of nanopure water. The pH of 0.5 M Tris base will beapproximately 10.8. Note that the Stop Reagent should be Tris base and not Tris-HCl. Store at room
temperature.

Storage and Stability•

Vivid® Substrates and Fluorescent Standards are stable for at least six months when stored desiccated andprotected from light at –20°C.

•

For long-term storage, reconstituted Vivid® Substrates and Fluorescent Standards should be kept
desiccated at –20°C. DMSO solutions are hygroscopic, and cold vials should be warmed to ambient
temperature before opening. After opening, they should be capped promptly to avoid reagent dilution byabsorbed moisture.

•

The CYP450 BACULOSOMES® Plus Reagent should be stored at –80°C.

•

The Vivid® Regeneration System should be stored at –80°C. Upon first thaw, aliquot into single use vials
as the reagent should not be subjected to additional freeze/thaw cycles.

•

The Vivid® NADP+ should be stored at –80°C and is stable for at least 10 freeze/thaw cycles. Store
protected from light.

•

The Vivid® CYP450 Reaction Buffer (2X) should be stored at room temperature.

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Vivid® CYP450 Screening Kits User Guide

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Assay Theory
Vivid® CYP450 Screening Kits are designed to assess metabolism and inhibition of human P450 isozymes involved inhepatic drug metabolism: CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2,
CYP3A4, and CYP3A5. The kits employ Vivid® Substrates and CYP450 BACULOSOMES® Plus Reagents. The CYP450
BACULOSOMES® Plus Reagents are microsomes prepared from insect cells expressing a human P450 isozyme, human
cytochrome P450 reductase, and in some cases, human cytochrome b5. CYP450 BACULOSOMES® Plus Reagents offer adistinct advantage over human liver microsomes in that only one CYP450 enzyme is expressed, thereby preventing
metabolism by other CYP450s. The Vivid® Substrates are metabolized by a specific CYP450 enzyme into products thatare highly fluorescent in aqueous solutions. Figure 1 schematically depicts the metabolism of a Vivid® Substrate into afluorescent metabolite. Note that the Vivid® Substrates have one or two potential sites for metabolism (indicated byarrows in Figure 1) and that oxidation at either site releases the highly fluorescent metabolite. For structures of theVivid® Substrates and Standards, visit us online at www.lifetechnologies.com/vivid.

Figure 1 Schematic of the metabolism of the “blocked” dye substrate into a fluorescent metabolite

The fluorescent metabolites are excited in the visible light spectrum, which minimizes interference caused by the
background fluorescence of UV-excitable compounds and NADPH. The excellent reaction kinetics and opticalproperties of the Vivid® Substrates allow their use at concentrations at or below their Km value in a reaction with P450
isozymes, assuring detection of even weak inhibitors and providing the convenience of room temperature or 37°C
incubations. The Vivid® CYP450 Assay may be run in a kinetic or endpoint mode (which is illustrated in Figure 2).

Figure 2 A schematic representation of an endpoint Vivid® CYP450 Assay

In end point mode, the test compounds (Step 1) are first combined with the Master Pre-mix (Step 2), consisting of
CYP450 BACULOSOMES® Plus Reagents and the Regeneration System (consisting of glucose-6-phosphate and glucose6-phosphate dehydrogenase). The Regeneration System converts NADP+ into NADPH, which is required to start the
CYP450 reaction. After a brief pre-incubation, the background fluorescence of the test compound and Master Pre-mix ismeasured (Step 3, Pre-read). The enzymatic reaction is initiated by the addition of a mix of NADP+ and the appropriate
Vivid® Substrate (Step 4) and plate is incubated for the desired reaction time. After the addition of a Stop Reagent(Step 5), the fluorescence is measured (Step 6).
In kinetic mode, the fluorescence is measured continuously starting after Step 4 (and eliminating Steps 5 and 6).
Standard curves, constructed from the supplied Fluorescent Standard, can be used to calculate reaction rates from theobserved fluorescence intensities in both assay formats. Assay parameters for all isoforms are listed in Tables 4 and 5(pages 8 and 9, respectively).
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Vivid® CYP450 Screening Kits User Guide

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Vivid® CYP450 High-Throughput Screening Assay Protocol
Each complete reaction must contain CYP450 BACULOSOMES® Plus Reagent, Vivid® Substrate, Vivid® NADP+, andVivid® Regeneration System in the appropriate Vivid® CYP450 Reaction Buffer. There are two possible modes for thisassay: kinetic and endpoint. The method you choose will depend on your analytical needs and the equipment
available.
•

The kinetic mode is useful for analysis of one multi-well plate at a time and does not require the additionof the stop reagent.

•

In endpoint mode, after an appropriate incubation time, the reaction is stopped by the addition of stop
solution. Endpoint mode allows the reaction to be performed in several multi-well plates simultaneously.

Note:

The following protocol is configured for use with one 96-well plate and 100 µL reactions. However, theprotocol can be modified to accommodate several different plate formats by adjusting the calculations forthe number of wells (and volume per well) in your experiment. Each kit supplies enough reagents for atleast 300 × 100 µL reactions.
Suggested assay conditions for screening with Vivid® kits are described in Table 1, below.

Table 1 Assay conditions
Condition

Test Compound

PurposeScreen for inhibition bycompound of interest

Positive Inhibition
Control

Inhibits the reaction with aknown P450 inhibitor andenables the subtraction of
background during data analysis

Solvent Control(No inhibitor)

Accounts for possible solvent
inhibition caused by introductionof test compounds originallydissolved in an organic solvent
such as DMSODispensing
40 µL 2.5X test compound
50 µL Master Pre-Mix
10 µL Vivid® Substrate and NADP+
40 µL 2.5X positive inhibition control (see page 12)
50 µL Master Pre-Mix
10 µL Vivid® Substrate and NADP+
40 µL 2.5X solvent control
50 µL Master Pre-Mix
10 µL Vivid® Substrate and NADP+

Thaw Reagents
1.

Place the CYP450 BACULOSOMES® Plus Reagent, Vivid® Regeneration System, and Vivid® NADP+ atroom temperature just until thawed (10–15 minutes), then store on ice until ready to use. Mix the CYP450
BACULOSOMES® Plus Reagent and Vivid® Regeneration System gently by inversion to obtain ahomogenous mixture after thawing. Do not vortex CYP450 BACULOSOMES® Plus Reagent or Vivid®
Regeneration System.

2.

Allow the Vivid® Substrate and Fluorescent Standard to warm to ambient room temperature(10–15 minutes) prior to reconstitution.

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Reconstitute Vivid® Substrate and Fluorescent Standard
1.

Reconstitute the Vivid® Substrate using anhydrous acetonitrile and the Fluorescent Standard using DMSO
(see Tables 2 and 3, below).

2.

Keep these solutions at room temperature for immediate use (≤ 4 hours), or store at -20°C.

Table 2 Reconstitution of the Vivid® Substrates
P450

Vivid® Substrate

Molecular
weightmg
per tubeµmol
per tube

1A2

Vivid® EOMCC

245.2

0.1

0.41

205

2

3

2A6

®

Vivid CC

171.2

0.5

2.92

290

10

10

2B6

®

Vivid BOMCC

307.3

0.1

0.32

160

2

3

2C8

Vivid® DBOMF

572.6

0.1

0.17

85

2

1

®

307.3

0.1

0.32

160

2

10

®

Vivid BOMF

452.5

0.1

0.22

110

2

1

Vivid® OOMR*

355.4

0.1

0.28

140

2

1

®

245.2

0.1

0.41

205

2

10

®

245.2

0.1

0.41

205

2

10

®

Vivid BOMCC
2C9
2C19

Vivid EOMCC
Vivid EOMCC

2D6

Vivid MOBFC

350.3

0.1

0.28

140

2

5

2E1

Vivid® EOMCC

245.2

0.1

0.41

205

2

10

2J2

Vivid® MOBFC

350.3

0.1

0.28

140

2

3

®

307.3

0.1

0.32

160

2

10

®

572.6

0.1

0.17

85

2

2

®

Vivid BOMCC
3A4

Vivid DBOMF

3A5*µL acetonitrile [stock solution] [screening concentration]
added per tube
(mM)
(µM)

Vivid BOMR

333.3

0.1

0.30

150

2

3

Vivid® BOMCC

307.3

0.1

0.32

160

2

10

Vivid® DBOMF

572.6

0.1

0.17

85

2

2

®

Heat Vivid OOMR Substrate at 50°C for 3–5 minutes and vortex to reconstitute.

Table 3 Reconstitution of the Fluorescent Standard. Use the blank cells in the table for your calculations. The value X is the amountof Standard listed on the tube label.
Assay Standardµmol per tube (X)Reconstitution SolventµL Reconstitution Solvent
added per tube (X x 10000)[Fluorescent Standard]
after reconstitution, µM

Example0.1

DMSO

1000 µL

100

Blue Standard

DMSO

100

Cyan Standard

DMSO

100

Green Standard

DMSO

100

Red Standard

DMSO/water (1:1)

100

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Vivid® CYP450 Screening Kits User Guide

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Prepare 1X Vivid® CYP450 Reaction Buffer
1.

Dilute 2X Vivid® CYP450 Reaction Buffer with nanopure water according to the Table 4, below. Thistypically provides enough 1X buffer for one 96-well plate; however, you can scale up or down thevolumes as needed. Use the diluted buffer for the preparation of standards, inhibitors, Master Pre-Mix,
and Vivid® Substrate/NADP+ solutions. Store the 1X buffer at room temperature.

Note:

The CYP2A6 assay requires Vivid® CYP450 Reaction Buffer II at 0.5X as its final concentration. All otherisoforms require the indicated buffer at 1X as the final concentration.

Table 4 Vivid® CYP450 Reaction Buffer Preparation
P450

Vivid® CYP450 Reaction Buffer

Concentration before
dilution

Desired concentration
Volume of buffer Volume of water
after dilution

1A2

Buffer I

200 mM

100 mM

10 mL

10 mL

2A6

Buffer II

100 mM

25 mM

5 mL

15 mL

2B6

Buffer I

200 mM

100 mM

10 mL

10 mL

2C8

Buffer II

100 mM

50 mM

10 mL

10 mL

2C9

Buffer II

100 mM

50 mM

10 mL

10 mL

2C19

Buffer II

100 mM

50 mM

10 mL

10 mL

2D6

Buffer I

200 mM

100 mM

10 mL

10 mL

2E1

Buffer III

400 mM

200 mM

10 mL

10 mL

2J2

Buffer II

100 mM

50 mM

10 mL

10 mL

3A4

Buffer I

200 mM

100 mM

10 mL

10 mL

3A5

Buffer I

200 mM

100 mM

10 mL

10 mL

Prepare Standard Curve (Optional)
We recommend that you use at least seven points (in addition to the blank) for the standard curve and perform it in
duplicate. The sample method below describes a seven-point standard curve set up in columns 1 and 2 of the 96-wellassay plate.
1.

Dilute the Vivid® Fluorescent Standard to 500 nM by mixing 5 µL of 100 µM Standard (Table 3, page 7)
with 995 µL of 1X Vivid® CYP450 Reaction Buffer.

2.

Add 200 µL of 500 nM Vivid® Fluorescent Standard to wells A1 and A2.

3.

Add 100 µL of 1X Vivid® CYP450 Reaction Buffer to each of the remaining wells in columns 1 and 2.

4.

Transfer 100 µL from wells A1 and A2 into the wells below (B1 and B2, respectively) containing 100 µL of
1X Vivid® CYP450 Reaction Buffer and mix by pipetting. This is a two-fold dilution.

5.

Repeat this dilution step, leaving wells H1 and H2 as an assay blank containing only 1X Vivid® CYP450
Reaction Buffer and no Standard. The resulting Fluorescent Standard concentrations are: 500 nM, 250 nM,
125 nM, 62.5 nM, 31.25 nM, 15.625 nM, 7.8125 nM, and 0 nM.

Note:

These are suggested initial concentrations for the standard curve. More or less may be appropriate
depending on your experimental needs.

Note:

The assay can be performed simply by using fluorescence values instead of converting to concentration of
product formed.

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Vivid® CYP450 Screening Kits User Guide

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Prepare Test Compounds, Positive Inhibition Control, and Solvent Control
1.

Prepare 2.5X Test Compounds by dilution into 1X Vivid® CYP450 Reaction Buffer.

2.

Prepare a 2.5X solution of a known P450 Inhibitor in 1X Vivid® CYP450 Reaction Buffer for positive
control of inhibition.

Note:

We recommend use of the inhibitors listed in Table 8, page 12.

3.

Prepare a solution of the solvent used to dissolve the test compounds and known P450 inhibitor at 2.5X
final concentration in 1X Vivid® CYP450 Reaction Buffer.

Note:

P450 activity can be greatly affected by organic solvents commonly used to dissolve test compounds. For
example solvent inhibition data, visit us online at www.lifetechnologies.com/vivid.

Dispense Test Compounds, Positive Inhibition Control, and Solvent Control
1.

Add 40 µL of the 2.5X solutions prepared above to desired wells of the plate.

2.

We recommend at least three replicates for the Positive Inhibition Control and Solvent Control.

Prepare and Dispense Master Pre-Mix1.

Prepare the Master Pre-Mix by diluting P450 BACULOSOMES® Plus Reagent and Vivid® Regeneration
System in 1X Vivid® CYP450 Reaction Buffer (see Table 5, below). Mix by inversion. Store this solution atroom temperature for immediate use.

Note:

Table 5, below, describes the preparation of Master Pre-mix for one entire 96-well plate. Depending on
your experimental needs, you may prepare more or less Master Pre-mix.

2.

Dispense 50 µL of Master Pre-Mix to each well.

Table 5 Master Pre-mix (mixture of CYP450 BACULOSOMES® Plus Reagent and Vivid® Regeneration System)
P450

Vivid Substrate

1A2

Vivid® EOMCC®

µL of Vivid®
µL of CYP450
Concentration ofµL of 1X Vivid® CYP450
Regeneration System BACULOSOMES® P450 in Master
Reaction Buffer added(100X) added
Plus added
Pre-mix (2X), nM

Screening
concentrationof P450, nM*

4850 (Buffer I)

100

50

10

5

2A6

®

Vivid CC

4800 (0.5X Buffer II)

100

100

20

10

2B6

®

4850 (Buffer I)

100

50

10

5

2C8
2C9

Vivid BOMCC®

Vivid DBOMF

4850 (Buffer II)

100

50

10

5

Vivid® BOMCC

4800 (Buffer II)

100

100

20

10

Vivid® BOMF

4800 (Buffer II)

100

100

20

10

®

4800 (Buffer II)

100

100

20

10

®

Vivid EOMCC

4850 (Buffer II)

100

50

10

5

Vivid® EOMCC

4800 (Buffer I)

100

100

20

10

Vivid® MOBFC

Vivid OOMR
2C19
2D6
2E1
2J2
3A4

4750 (Buffer I)

100

150

30

15

®

4700 (Buffer III)

100

100

40

20

®

Vivid MOBFC

4850 (Buffer II)

100

50

10

5

Vivid® BOMCC

4850 (Buffer I)

100

50

10

5

Vivid® DBOMF

4850 (Buffer I)

100

50

10

5

Vivid BOMR

4850 (Buffer I)

100

50

10

5

Vivid® BOMCC

4850 (Buffer I)

100

50

10

5

4850 (Buffer I)

100

50

10

5

Vivid EOMCC

®

3A5

®

Vivid DBOMF*For your first experiment, we suggest these concentrations of P450. Based on your results, you may find more or less enzyme isnecessary.
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Page 10 of 13

Pre-Incubate
1

Incubate the plate for 10 minutes at room temperature to allow the compounds to interact with the P450 in
the absence of enzyme turnover.

2.

During this pre-incubation, prepare a 10X mixture of Vivid® Substrate and Vivid® NADP+ (Table 6, below).

Note:

Table 6, below, describes the preparation of a Vivid® Substrate and Vivid® NADP+ mixture for one entire
96-well plate. Depending on your experimental needs, you may prepare more or less of this reagent.

3.

You may also wish to include a pre-read at this point to determine if your compounds are fluorescent.

Table 6 Vivid® Substrate and Vivid® NADP+ MixtureµL of 1X Vivid® CYP450
Reaction Buffer addedµL of Vivid®
µL of Reconstituted[Vivid® Substrate] in [Vivid® NADP+] in
NADP+ (100X)
Substrate added*
10X mixture (µM) 10X mixture (µM)
added

P450

Vivid Substrate

1A2

Vivid® EOMCC

955 (Buffer I)

15

30

30

300

2A6

Vivid® CC

960 (0.5X Buffer II)

10

30

100

300

2B6

®

955 (Buffer I)

15

30

30

300

®

895 (Buffer II)

5

100

10

1000

®

Vivid BOMCC

920 (Buffer II)

50

30

100

300

Vivid® BOMF

895 (Buffer II)

5

100

10

1000

Vivid® OOMR

895 (Buffer II)

5

100

10

1000

Vivid EOMCC

920 (Buffer II)

50

30

100

300

Vivid® EOMCC

920 (Buffer I)

50

30

100

300

®

945 (Buffer I)

25

30

50

300

®

920 (Buffer III)

50

30

100

300

®

Vivid MOBFC

955 (Buffer II)

15

30

30

300

Vivid® BOMCC

920 (Buffer I)

50

30

100

300

®

890 (Buffer I)

10

100

20

1000

®

Vivid BOMR

885 (Buffer I)

15

100

30

1000

®

Vivid BOMCC

920 (Buffer I)

50

30

100

300

Vivid® DBOMF

890 (Buffer I)

10

100

20

1000

2C8
2C9
2C19
2D6
2E1
2J2
3A4

3A5

®

Vivid BOMCC
Vivid DBOMF®

Vivid MOBFC
Vivid EOMCC

Vivid DBOMF*See Reconstitute Vivid® Substrate and Fluorescent Standard, page 7.

Start Reaction
1.

Start the reaction by adding 10 µL per well of the 10X Vivid® Substrate and NADP+ mixture prepared in
Pre-Incubation step (Table 6, page 10).

Measure Fluorescence
1.

Kinetic Assay Mode (recommended): Immediately (less than 2 minutes) transfer the plate into thefluorescent plate reader and monitor fluorescence over time at excitation and emission wavelengths listed
in Table 7, page 11. Initially, we recommend reads in 1-minute intervals for 60 minutes. Based on these
initial results, you can further refine the measurement times for subsequent assays, as desired.
Appropriate reaction times will vary by kit and experimental conditions.

2.

Endpoint Assay Mode: Incubate the plate for the desired amount of time, and then add 50 µL of Stop
Reagent (0.5 M Tris base) to each well to quench the reaction. Measure fluorescence in the fluorescent plate
reader at excitation and emission wavelengths listed in Table 7, page 11.

3.

Proceed to data analysis, page 11.
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Vivid® CYP450 Screening Kits User Guide

Page 11 of 13

Table 7 Recommended excitation and emission wavelengths and filter sets
Vivid® Fluorescent Standard*
Red
Fluorescence
Plate Reader
monochromatorfilter-based

Blue

Green

Excitation/
Emission

center
(nm)

Band
width

center
(nm)

Band
width

excitation

550

12

415

emission

590

12

460

excitation

535

25

emission

590

20

Cyan

center
(nm)

Band
width

center
(nm)

Band
width

20

490

20

520

12

415

20

12

520

20

415

20

485

20

415

20

460

20

520

25

520

25

*Red Standard is sodium salt of resorufin. Green Standard is fluorescein. Blue Standard is 3-cyano-7-hydroxycoumarin. Cyan
Standard is 7-hydroxy-4-trifluoromethylcoumarin. We recommend exciting the Cyan and Blue Standards off-peak (>400 nm) to
minimize background from NADPH fluorescence.

Suggested Protocol for the Analysis of Results
Kinetic Assay Mode
1.

Obtain reaction rates by calculating the change in fluorescence per unit time.

2.

Calculate the percent inhibition due to presence of test compound or positive inhibition control using theequation:where X is the rate observed in the presence of test compound, A is the rate observed in the absence of
inhibitor (solvent control or no inhibitor control, as appropriate), and B is the rate observed in the presenceof the positive inhibition control.

Endpoint Assay Mode1.

Calculate percent inhibition due to presence of test compound or positive inhibition using the following
equation:where X is the fluorescence intensity observed in the presence of test compound, A is the fluorescenceintensity observed in the absence of inhibitor (solvent control or no inhibitor control, as appropriate), andB is the fluorescence intensity observed in the presence of the positive inhibition control.
Optional:

Both types of data analysis above can be performed using a standard curve (see Prepare Standard Curve,
page 8) to calculate the reaction rates as nmol product formed per unit time, if desired.

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Vivid® CYP450 Screening Kits User Guide

Page 12 of 13

Suggested Inhibitors
Table 8 Suggested Inhibitors
EnzymeInhibitor

Recommended Vendor (Cat. no.)Suggested Final
Concentration (1X)

CYP1A2α-naphthoflavone

Sigma-Aldrich (N5757)

10 µM

CYP2A6

tranylcypromine

Sigma-Aldrich (P8511)

100 µM

CYP2B6

miconazole

Sigma-Aldrich (M3512)

30 µM

CYP2C8montelukast

Cayman Chemical (10008318)

10 µM

CYP2C9sulfaphenazole

Sigma-Aldrich (S0758)

30 µM

CYP2C19

miconazole

Sigma-Aldrich (M3512)

30 µM

CYP2D6quinidine

Sigma-Aldrich (Q3625)

10 µM

CYP2E1tranylcypromine

Sigma-Aldrich (P8511)

1 mM

CYP2J2

terfenadine

Sigma-Aldrich (T9652)

25 µM

CYP3A4ketoconazole

Sigma-Aldrich (K1003)

10 µM

CYP3A5ketoconazole

Sigma-Aldrich (K1003)

10 µM

References
Cohen, L.H, et al. (2003) In vitro drug interactions of cytochrome P450: an evaluation of fluorogenic to conventional
substrates. Drug Metab. Dispos. 31:1005-15.
Marks, B.D., et al. (2002) A high throughput screening assay to screen for CYP2E1 metabolism and inhibition using afluorogenic Vivid® P450 substrate. Assay Drug Dev. Technol. 1:73-81.
Marks, B.D., et al. (2003) High-throughput screening assays for CYP2B6 metabolism and inhibition using fluorogenic
Vivid® substrates. AAPS PharmSci. 5:E18.
Marks, B.D, et al. (2004) High-throughput screening assays for the assessment of CYP2C9*1, CYP2C9*2, and CYP2C9*3
metabolism using fluorogenic Vivid® substrates. J. Biomol. Screen. 9:439-49.
Trubetskoy, O.V., et al. (2005) Highly miniaturized formats for in vitro drug metabolism assays using Vivid®
fluorescent substrates and recombinant human cytochrome P450 enzymes. J. Biomol. Screen. 10:56-66.

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Vivid® CYP450 Screening Kits User Guide

Page 13 of 13

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