Category: Assay Development and Screening

1152-C - A New Robust Fluorescent Calcium (Ca2+) Indicator for Ca2+ Flux Assays in Challenging GPCR Targets

Tuesday, February 6, 2018
2:00 PM - 3:00 PM

 Measurement of intracellular Ca2+ is widely used in characterizing G protein–coupled receptors (GPCRs) agonists and antagonists in drug discovery, as well as in monitoring synaptic activities in neuroscience research.  Fluorescent Ca2+ indicators (e.g. Indo-1, Fura-2, Fluo-3 and Fluo-4) are commonly used to monitor Ca2+ response in a variety of applications including HTS, fluorescent imaging and flow cytometry.   However, these dyes require the presence of an organic anion transporter inhibitor (e.g. probenecid) to prevent the leakage of the indicators in many cell types (e.g. CHO, Hela). The organic anion transporter inhibitors are toxic to cells and some of them are known to be the inhibitors of certain GPCRs (e.g. chemokine receptors, bitter taste receptors). Because of those reasons, the usages of these dyes are limited.  


We have recently developed the first probenecid-free fluorescent Ca2+ indicator, Calbryte 520 AM. Calbryte 520 AM is cell permeable and non-fluorescent. When binding to Ca2+, Calbryte 520 produces exceptionally bright fluorescent signal that can be detected at Ex/Em =490 nm/525 nm and very high signal to background (S/B) ratio (10 fold).


In this study, Calbryte 520 AM and Fluo-4 AM were used to determine the EC50 of M1 Muscarinic Receptor agonist carbachol and antagonist atropine in CHO-M1 cells, the EC50 of C-C Chemokine Receptor Type 5 (CCR5) ligand RANT in CHO-CCR5 cells and the EC50 of P2Y receptor agonist ATP in CHO-K1 cells with or without probenecid incubation. The Ca2+ flux induced fluorescent signal change was recorded using fluorescent microplate reader FlexStation, fluorescent microscope and Flow Cytometer. Without probenecid incubation, only Calbryte 520 AM captured the change of fluorescent signal due to Ca2+ flux inside the cells. With probenecid incubation, when intracellular Ca2+ releaseoccurred, Calbryte 520 AM incubated cells exhibited 5-10 times brighter signal and 2-5 fold higher S/B ratio than cells incubated with Fluo-4 AM.  Without probenecid incubation, the agonists and antagonists also showed more sensitive dose dependent response, which is demonstrated by 2-3 fold lower EC50.  In the capacitive calcium entry assay, upon concanavalin A stimulation, CHO cells incubated with Calbryte 520 AM and probenecid showed only 2 fold Ca2+ response instead of 6 fold response in cells incubated with Calbryte 520 AM alone. These results demonstrated the advantage of using a probenecid free assay condition.


In conclusion, the innovative Calbryte 520 AM Ca2+ indicator enables probenecid free assay for monitoring Ca2+ mobility with its much improved cellular retention, brightness and S/B ratio.

Muhua Yang

Scientist
AAT Bioquest
Sunnyvale, CA

Scientist, 2017- Present
AAT Bioquest, Biology Department (Sunnyvale, CA)
• Product application development and support for chemical products designed for industrial and academic biomedical research
Scientist, 2016 – 2017
WuXi Apptec, Research Science Division, In Vitro Biology (Plainsboro, NJ)
• Assay development and HTS using automation technology for client based research
Project Leader/Research Scientist, 2011 – 2016
Venenum Biodesign, Institute of Metabolic Disorders (Hamilton, NJ)
• Management and execution of early drug discovery programs in metabolic disorder
Postdoctoral Fellow, 2010 – 2011
University of Maryland, School of Medicine (Baltimore, MD)
• Study of epigenetic regulation of key molecules in human breast cancer Initiation, progression and prevention.
Ph. D. Biological Sciences, 2004-2010
University of Delaware, Biological Sciences Department, Cell and Organ System Physiology (Newark, DE)
B.S. Medicine, 1997-2002
Peking University, Health Science Center (Beijing, P.R. China)