Why is my Fluo-4 signal not at a useful magnitude?

If using an in-house procedure, try a Calcium assay kit suitable for your needs to investigate if the procedure or the material is the cause.

High indicator concentrations can damage cells or have a Ca²⁺quenching effect. Test different concentrations for efficacy and use the lowest molarity possible.

Unusable fluorescent signal indicates that the Kd value (dissociation constant) for the chosen fluorophore isn't suitable. Fluo-4 has a Kd value of 345 nM. Use an alternative such as Calbryte™ 520 AM, with a Kd value of 1200 nM, or Fluo-8FF™ AM, with a Kd value of 10,000 nM.