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The main steps for the FLUOFORTE® Calcium assay kit (ENZ51016) involves loading cells with dye that fluoresce when exposed to free calcium. Once the cells are treated with the desired test agent, calcium will be released from the endoplasmic reticulum. Free (mobilized) calcium then binds the preloaded dye and the resulting transient fluorescence change can be detected and quantified in real time by flow cytometry, fluorescence microscopy of microplate reader.