Bioluminescent Proteins: High Sensitive Optical Reporters for
Novel Bioluminescent Cell Metabolism Assay Integration ... · IC 50 = 70nM 8hr. 24hr. 48hr. 72hr....
Transcript of Novel Bioluminescent Cell Metabolism Assay Integration ... · IC 50 = 70nM 8hr. 24hr. 48hr. 72hr....
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