Date www.promega.com

Novel Bioluminescent Cell Metabolism Assays Integration with HP D300 Digital Dispenser and Tecan Gas Controlled Module Equipped Infinite M200 Pro Reader.

Gediminas Vidugiris1, Donna Leippe1, Mary Sobol1, Sarah Duellman1, Wenhui Zhou2, Jolanta Vidugiriene1,

Jessica Merlino3, Michael Reitman4 Thomas Hengstl5, Poncho Meisenheimer2, Cristopher Cowan1, James Cali1

1Promega Corporation, Madison, WI; 2Promega Biosciences, LLC, San Luis Obispo, CA; 3Tecan Schweiz, Männedorf, Switzerland; 4Tecan US, Morrisville, NC; 5Tecan Austria GmbH, Groedig, Austria.

1. Introduction 4. Bioluminescent NAD, NADH, NADP and NADPH Detection 7. Automation And Multiplexing: Results

2. Advantages Offered by Live Cell Bioluminescence Viability Assay 5. Advantages: Ease of Use with Improved Sensitive and Robustness 8. Advantages Offered by Digital Dispenser

3. Continuous monitoring of drug induced changes in cell viability 6. Automation And Multiplexing: Experimental Outline 9. Conclusions

Robustness of bioluminescence assays makes them attractive and easily adoptable for automation and HTS

applications. We describe the integration of Tecan, Hewlett Packard instrumentation capabilities with two of Promega’s

recently developed assays targeting cell metabolism applications:

1) Bioluminescence Live Cell Viability Assay and 2) NAD(P)/NAD(P)H Detection Assays.

The luminescence signal was determined every hour for 72 h in an Infinite M200 reader with GCM

IC50 values were calculated at indicated time points

Plate K562 cells (2K cells/30ul) containing Cell Viability

Reagents added to the media (by Thermo Combi_nL)

Place in M200 multimode reader

Read luminescence every hour

Add inhibitors using HP D300 Dispenser

Bioluminescence Live Cell Viability Assay allows continuous monitoring of changes in viability in real time

Tecan’s Infinite M200 PRO multimode reader with Gas Control Module (GCM) enables long-term kinetic measurements

Time dependent changes in cell viability and IC50 values are determined from the same well

Assay principal: Measures the reducing capacity of metabolically active cells

Allows measurement of changes in real time

S/B>120

S/B>5

AIM: Evaluate effect of Rotenone - an electron transport chain inhibitor and FK866 - a known inhibitor of NAD

biosynthetic pathway, on cell viability and NAD/NADH levels

Read luminescence to determine the amount of viable cells/well to

control for plating reproducibility

Add 30ul NAD/NADH Detection Reagent

Read luminescence

Incubate 24h

Time dependent decrease in cell viability

No decrease in cell viability A novel proluciferin substrate for diaphorase enzyme was combined with NAD or NADP specific cycling enzymes to

develop three assay formulations

Measures reduced

Measures phosphorylated Measures non phosphorylated

The most sensitive assay in the market Wide assay window

Detects changes directly in cell lysate

Log(FK866), nM

-2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5

% C

on

tro

l

0

20

40

60

80

100

120 IC50 = 0.3nM

Assay Measures LOD,

nM

Linear

range

S/B Z’

NADH/NADPH-

Glo

NADH and/or

NADPH 25-50

25nM-

25uM ~500

>0.8 at

0.5uM

NAD/NADH-Glo NAD + NADH,

Only NAD

Only NADH

0.25-1 0.5nM-

0.5uM ~400

>0.8 at

200nM

NADP/NADPH-

Glo

NADP + NADPH

Only NADP

Only NAPH

0.25-1 0.5nM-

0.5uM ~400

>0.8 at

200nM

Two novel bioluminescence approaches were developed to monitor cellular metabolism pathways

Live cell viability assay is based on measuring the metabolic activity of live cells

A novel bioluminescent sensing system is added directly into cell culture medium

The luminescence output generated by live cells is correlated with the amount of viable cells

The assay is sensitive (detects less than 10 cells/well) and has a wide dynamic range (S/B>100)

The unique feature of the assay is the ability to continuously monitor changes in cell viability in real time with

multiple measurements performed from the same well

Tecan’s Infinite M200 PRO multimode reader with Gas Control Module (GCM) enables long-term kinetic

measurements and takes full advantage of the capabilities offered by the live cell viability assay

To address the need for rapid and robust measurement of adenine dinucleotides, three assay formats

were developed based on the use of a novel proluciferin diaphorase substrate:

The assays are easy to use (add and read) and are amenable to HTS screening (Z’>0.8)

The sensitivity (LOD~1nM) and large signal window (S/B>300) of the assays enable measurement of the

changes in NAD(P)/NAD(P)H levels in a homogenous format directly in 96 and 384 well plates

The HP D300 Digital Dispenser, available from Tecan, provided rapid and convenient dispensing of

compounds and was successfully applied for assay validations

FK866 inhibitor was dispensed to K562 cells plated in 384 well plates using four different methods: two on HP D-300,

manual and robotic on Hudson Robotics Solo.

Total NAD/NADH was measured using NAD/NADH-Glo assay and results were evaluated by comparing dispensing

reproducibility (%CVs) and calculating EC50 values.

Advantages using HP D-300

Direct Titrations

•Better results reproducibility - %CV:

•Significant time savings preparing one

384 plate by direct titration ~2.5min.

vs. ~10 min. by Serial Dilutions.

•Dispense any dose to any well. Finely

targeted and spaced doses results in

more precise EC50 determination.

•Titrations normalized to DMSO.

•Fast and intuitive programing process.

•Preserve compounds - dead Vol.<2μl.

A B

D C

HP D300 Dispenser

Non-contact, direct

titration of compounds

Broad volume range:

13 pl up to 10 µl

Low Dead Volume ~2 µl

Efficient experiment

design

Dispense any dose to

any well

Save time and lab ware

Live Cell Bioluminescence Viability Assay

NAD, NADH, NADP, NADPH Bioluminescence Assays

Multiplexing

Rigorous environmental

control inside Infinite®

200 PRO multimode

reader

Control of O2 and CO2

concentrations

Top or bottom

microplate reading

Advanced temperature

control and automated

Z-focusing

Gas Control Module

%RStDev (%CV) 3.2 2.7 4.4 4.9

EC50 0.70 0.79 0.71 0.62

D-300

Variable

Ser. Dil.

Solo-8

Manual

Ser. Dil.

4 replicates at

each [c] (n=4 )

D-300

Linear

Log(Digitonin), ug/ml

-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5

% C

on

tro

l

0

20

40

60

80

100

120

Lum

AlamBlue

AlamBlue-bc

Lum

IC50 = 20ug/ml

S/B=67

AlamarBlue

IC50=18.7ug/ml

S/B= 2.3

Rapid decrease in NAD/NADH levels

Slight decrease in NAD/NADH levels

NADP

NADPH NAD

NADH

NADH

NADPH

Increased sensitivity and assay window

o Samples (21 columns x 16 rows)

nM

nM Log(Rotenone), nM

-1 0 1 2 3

% C

on

tro

l

0

20

40

60

80

100

120

IC50 = 70nM

8hr.

24hr.

48hr.

72hr.

nM