Development of an aequorin luminescence calcium assay for high-throughput screening using a plate reader, the LumiLux.
A luminescence assay using a new plate reader, the LumiLux (PerkinElmer, Waltham, MA), has been validated for high-throughput screening (HTS). In this study, we compared the aequorin luminescence-based calcium mobilization assay to the fluorescence-based calcium assay. A cell line stably co-expressing apo-aequorin, a chimeric G-protein, and a G-protein-coupled dopamine receptor was used to screen a collection of 8,106 compounds using the Hamamatsu Photonics (Bridgewater, NJ) FDSS6000 and LumiLux as the plate readers. The assay parameters evaluated included hit rate correlation, signal-to-noise ratio, and overall assay performance calculated by Z and standard deviation. The average Z values and hit rates were comparable between assay platforms;however, the standard deviation for the agonist aequorin assay was significantly smaller. There was also a significant decrease in the number of false-positives with the aequorin assay. These results suggest that the aequorin assay in combination with the new plate reader, LumiLux, provides a simple, cost-effective, robust, and sensitive assay for HTS
Menon
Veena
V
High-Throughput Screening, Biological Research, Lundbeck Research USA, Inc. 215 College Road, Paramus, NJ 07652, USA.
Ranganathn
Arun
A
Jorgensen
Vincent H
VH
Sabio
Michael
M
Christoffersen
Claus T
CT
Uberti
Michelle A
MA
Jones
Kenneth A
KA
Babu
Poda Suresh
PS
D000331:Aequorin / Q000737:chemistry*; D000814:Aniline Compounds / Q000737:chemistry; D000818:Animals; D001059:Apoproteins / Q000032:analysis / Q000737:chemistry; D016466:CHO Cells; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D016000:Cluster Analysis; D006224:Cricetinae; D003412:Cricetulus; D018492:Dopamine Antagonists / Q000032:analysis / Q000145:classification / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000191:economics / Q000379:methods*; D005189:False Positive Reactions; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D007093:Imidazoles / Q000737:chemistry; D020128:Inhibitory Concentration 50; D007700:Kinetics; D049408:Luminescent Agents / Q000737:chemistry*; D008163:Luminescent Measurements / Q000191:economics / Q000379:methods*; D011719:Pyrazines / Q000737:chemistry; D044169:Receptors, Calcium-Sensing / Q000032:analysis* / Q000378:metabolism; D011954:Receptors, Dopamine; D012371:Robotics / Q000191:economics; D012984:Software; D014966:Xanthenes / Q000737:chemistry
D000814:Aniline Compounds; D001059:Apoproteins; D018492:Dopamine Antagonists; C409648:Fluo 4; D005456:Fluorescent Dyes; D007093:Imidazoles; D049408:Luminescent Agents; D011719:Pyrazines; D044169:Receptors, Calcium-Sensing; D011954:Receptors, Dopamine; D014966:Xanthenes; C017144:coelenterazine; D000331:Aequorin; D002118:Calcium