Intracellular Calcium measurement with Fluo-8

G Protein Coupled Receptors (GPCR) and ion channels are important targets in drug development. Both target classes represent membrane proteins which require convenient and robust cell-based assays for screening campaigns. Usually used as readout of such screenings, intracellular Calcium concentration implies the use of Ca2+ indicators able to meet demanding expectations of researchers engaged in assay development and screenings.

Measurement of intracellular Calcium  based on fluorescent dyes

intracellular calcium measurement with Fluo-8

Chemical structure of Fluo-8

Commonly used tools to directly measure Calcium are Ca2+ chelators – in this case fluorescent dyes which directly bind Ca2+. Upon binding, the fluoresence intensity is significantly increased and can be easily measured by fluorimetric instruments. Classical dyes are Fura-2, Indo-1, Fluo-3, Fluo-4.

Fluo-8 (chemical structure, see Fig 1) can be considered as the state-of the-art indicator for measuring intracellular Ca2+ concentration, as it has 3 main benefits over classical fluorescent dyes:

  1. Twice as bright as Fluo-4 and four time brighter than Fluo-3
  2. Loading of the cell can be conducted at room temperature, meaning under less harsh conditions than with Fluo-3 and Fluo-4 which require 37°C for optimal loading.
  3. As especially HTS screening requires homogeneous assays (assays not requiring any wash steps to be HTS compatible), a Fluo-8 no wash kit is available as well.

    Fluo-8 outperforms Fluo-4 in measuring Intracellular calcium

    Fluo-8 dye is twice brigther than Fluo-4.

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Intracellular calcium measurement: Fluo-8 dye compared with Fluo-3 and Fluo-4 dyes

Rat embryo fibroblasts before and after stimulation with 2 µM Ionomycin and loaded with Fluo-3, Fluo-4, or Fluo-8.


Written by Ali El Baya, PhD
Ali el Bayâ part of our Customer Success Team.