Gen5 version required: 1.0 or higher
Basis for the Assay:
Detection of Calcium (Ca+2) ions is most often accomplished by using sensing probes or dyes. These dyes typically consist of a fluorophore and a region that confers analyte specificity, such a BAPTA group for Ca+2. The multiple anionic carboxyl groups bind the divalent calcium ion in a similar fashion as the calcium specific chelator, ethylene glycol bis(β-aminoethylether ether)-N,N,N’,N’-tetraacetic acid (EGTA). The indicator dye Calcium Green increases its fluorescence in the presence of calcium. The increase in fluorescence is proportional to the calcium ion concentration.
The protocol calls for a fluorescence measurement using a 485/20 excitation filter and a 528/20 emission filter.
The experiment data file contains all of the fluorescence determinations for a calcium determination using calcium green dye.
Plate configuration assumes that samples will be run in duplicate. Included is a standard curve of calcium concentrations (0 to 39 μM) available from Molecular Probes’ (Invitrogen) calcium calibrator kit. There are no blanks or controls. Thus 37 samples can be run on a microplate in duplicate in addition to the standard curve.
The report is configured to provide the (1) raw fluorescence, (2) standard curve, and (3) calculated calcium concentrations of unknown samples.