HitHunter® Calcium No WashPLUS Assay Platform

DiscoverX’s HitHunter Calcium No WashPLUS Assay is validated for use with all Gq-coupled cell lines. This fluorescent-based assay uses a homogenous assay format to detect calcium mobilization and is compatible with adherent or suspension cells, GPCRs, and calcium channels. Measurement of intracellular calcium provides valuable information on the activation status of GPCRs and ion channels. After loading the cells with the fluorescent dye, stimulation of the receptor or calcium channel releases intracellular calcium, causing the calcium-sensitive dye to fluoresce. The Calcium No WashPLUS Assay provides a fast, reliable, and low-cost alternative to traditional wash and quench-based assays.

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Learn more about the updated Cell Culture and Handling Procedure in the new Technical Bulletin.

HitHunter Calcium No WashPLUS Assay Principle


HitHunter Calcium No WashPLUS Assay Principle.  The esterified (inactive) calcium dye (probe) penetrates the cell membrane and becomes active once inside the cell.  The active form of the dye becomes fluorescent after binding to intracellular calcium. Additive A is added to prevent the dye from being released out from the cell. Ligand binding stimulates GPCR activation, resulting in release of intracellular calcium stores from the ER, which leads to an increase in fluorescence in the presence of the activated calcium dye.


To view the available calcium assay products, please check out the links below.
HitHunter Calcium No WashPLUS  Assay
Coelenterazine H -- All aequorin-based luminescence assays require addition of a coelenterazine substrate. DiscoverX offers an optimized form called Coelenterazine H. This derivative of native coelenterazine is significantly more sensitive to calcium and provides enhanced performance in aequorin-based luminescent HTS calcium assays.

Related Products and Services

Calcium Gq-Coupled Cell Lines
Ligands, Inhibitors, and Agonists
HitHunter cAMP Assays
PathHunter® β-Arrestin Assays


GPCR Profiling
Custom Assay Development


HitHunter Calcium No WashPLUS Assay Highlights

  • Single step, HTS friendly no wash protocol assay to use with native Gq-coupled PathHunter® cell lines
  • Large assay windows, larger dynamic range, and  improved signal and performance over FLUO-3
  • Multiplex with β-Arrestin recruitment using the same cell line and the same well for multiple readouts

Uncover Unique Pharmacologies, Rank-Order Leads, and Analyze Calcium Flux

Potency and rank order of five Histamine H1 receptor antagonists as assessed by calcium signaling using the HitHunter Calcium No WashPLUS kit.  Rank order is consistent with affinity data reported for these antagonists in the IUPHAR database. Data kindly provided by Dr. Thierry Calmels at BioProjet Biotech, France.

The small molecule MDL29,951 induces a concentration-dependent agonistic response in calcium signaling after binding to the orphan GPCR, GPR17.

Calcium and ß-Arrestin Detection in the Same Well

DiscoverX offers Gq-coupled β-arrestin targets as clonal cell lines or ready-to-use kits, to monitor intracellular calcium flux in real time, followed by the recruitment of β-arrestin in the same cell line or in a single well. This HTS-compatible duplexed assay increases your screening capabilities, removes challenges of running multiple assays, reduces data complexity, and provides significant cost savings and time efficiencies.

Naturally Gq-coupled PathHunter β-arrestin cell line expressing the Bradykinin B1 Receptor (BDKRB1) was first monitored for calcium mobilization using the HitHunter Calcium No WashPLUS Kit as a fluorescent readout. β-Arrestin recruitment was then analyzed in the same well using PathHunter Detection Reagents. This duplexed assay provides a simple and efficient approach for measuring different signaling pathways for the same GPCR and defining potency ranking of GPCR modulators.