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HitHunter® Calcium No WashPLUS Assay Platform

DiscoverX’s HitHunter Calcium No WashPLUS kits use a homogenous assay format to detect calcium mobilization. Measurement of intracellular calcium provides valuable information on the activation status of GPCRs and ion channels. Stimulated receptor releases intracellular calcium, causing the calcium-sensitive dye in the kit to fluoresce. Our calcium assay kits provide a fast, reliable, 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 Assay Principle

calcium nowash

HitHunter® Calcium No WashPLUS Assay Principle.  The esterified (inactive) calcium dye (probe) penetrates through the cell membrane and becomes active within the cell.  Once the dye is active, the dye binds to intracellular calcium and fluoresces. Additive A is added to prevent the dye from being released from the cell. So upon ligand stimulation, the receptor signals release of intracellular calcium, which results in an increase of dye fluorescence.

Aequorin-based Calcium Assays

All aequorin-based luminescence assays require addition of a coelenterazine substrate. DiscoverX offers an optimized form called Coelenterazine H (90-0084).  This derivative of native coelenterazine is significantly more sensitive to calcium and provides enhanced performance in aequorin-based luminescent HTS calcium assays.

Calcium Flux Analysis Using HitHunter® Calcium No WashPLUS Assay

Adrenergic alpha 1B receptor     

   

EDG3   

  cGMP-PDE-V-Inhibition-curve-data
Uncover unique pharmacologies for all classes of compounds. Depicted are dose curves identifying responses to various agonists for the Adrenergic alpha 1B receptor [click graph to enlarge].
 
EDG3 cells (squares) and native parental cells (triangles) were stimulated with a control agonist and calcium influx was detected with FLIPR® Tetra [click graph to enlarge]

Features & Benefits

  • Single step, no wash protocol
  • Large assay windows, larger dynamic range and  improved signal & performance over FLUO-3
  • Flexible protocol - use with adherent or non-adherent cells, GPCRs & calcium channels
  • Fewer false positives, due to reduced spontaneous calcium flux and minimal perturbation of cells
  • Validated for use with all Gq-coupled PathHunter® cell lines