Compound Interference of CellTiter-Glo® vs PE ATPlite™ 1 step
Brad Hook and Trista Schagat
Promega Corporation 2800 Woods Hollow Road, Madison, WI U.S.A. 53711
3. ATP addition – CellTiter-Glo® is more resistant to
inhibitors than PE ATPlite™ 1step
1. Abstract / Introduction
Blue-PE ATPlite™ 1step
RED-CellTiter-Glo®
RED-CellTiter-Glo®
120
Blue-PE ATPlite™ 1step
GREEN-BacTiter-Glo®
120
100
100
80
80
% Activity
60
40
60
40
20
20
0
-8
-7
-6
-5
-4
-3
Log [Compound], M
100
CTG Substrate
90
BTG Substrate
80
PE Substrate
70
% Activity
% Activity
Reducing the number of false positives in a high-throughput screen is a key
goal for any researcher trying to minimize downstream efforts. Luciferasebased systems are used widely as reporters for drug screens; however,
compounds in a library can inhibit luciferase, resulting in false
positives. Ultra-Glo™ rLuciferase, an evolved luciferase from the firefly,
Photuris pennsylvanica, is less sensitive to compound inhibition than wildtype firefly luciferase. In this study, we directly compare the abilities of the
Promega CellTiter-Glo® Luminescent Cell Viability Assay, an Ultra-Glo™
rLuciferase-based luminescent ATP detection system, and the Perkin Elmer
ATPlite™ 1step assay to resist common commercial luciferase
inhibitors. The CellTiter-Glo® Assay has greater than 80% activity for 5 out of
the 7 compounds at an inhibitor concentration of 10µM; whereas, ATPlite™
1step has 80% activity for only 1 out of 7 compounds at the same inhibitor
concentrations.
6. BacTiter-Glo® and CellTiter-Glo® have same
resistance to compound inhibitors
0
-7
-6.5
-6
-5.5
-5
-4.5
Log [Compound], M
-4
-3.5
-3
60
50
40
30
20
10
0
2. Materials and Methods
Seven known luciferase inhibitors were mixed with ATP detection
reagent followed by addition of either ATP or mammalian cells.
Luminescence was measured on the GloMax®-Multi+ Detection System.
At the common compound concentration of 10µM, CellTiter-Glo® has
greater than 80% activity for 5 out of the 7 compounds; whereas ATPlite™
1step has 80% activity for only 1 out of 7 compounds
4. Mammalian cell addition – CellTiter-Glo® is more
resistant than PE ATPlite™ 1step
RED-CellTiter-Glo®
Blue-PE ATPlite™ 1step
7. Summary
The Ultra-Glo™ rluciferase in the CellTiter-Glo® Assay exhibited less
compound interference when used as an ATP sensor compared to the lucPpy
in the Perkin Elmer ATPlite™ 1step assay. When performing drug screening
activities, Ultra-Glo™-based luciferase systems will result in lower false
positives because of this increased resistance to compound interference.
120
100
80
% Activity
Both BacTiter-Glo® and CellTiter-Glo® contain Ultra-Glo™ rluciferase and
both have similar resistance to luciferase inhibitors.
60
40
20
0
-7
-6.5
-6
-5.5
-5
-4.5
-4
-3.5
-3
Log [Compound], M
REFERENCES
Auld, D.S. et al. (2008) Characterization of chemical libraries for luciferase inhibitory activity. J. Med. Chem. 51, 2372–86.
Auld, D.S. et al. (2009) A basis for reduced chemical library inhibition of firefly luciferase obtained from directed evolution. J. Med. Chem. 52, 1450–8.
At 20,000 cells per reaction with compound concentrations of 10µM,
CellTiter-Glo® has greater than 80% activity for 5 out of the 7 compounds;
whereas ATPlite™ 1step has 80% activity for only 1 out of 7 compounds
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