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Why is my Fluo-4 signal not at a useful magnitude?
Posted February 19, 2020
Answer
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.
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