Gen5 version required: 1.0 or higher
Basis for the Assay:
Cholesterol oxidase reacts with D-glucose to form D-gluconolactone and hydrogen peroxide (H2O2). Amplex red reagent and H2O2 are converted to resorufin and water in a one-to-one stiochiometry by horseradish peroxidase. Resorufin is a highly fluorescent compound with absorption maxima of 571 nm and a peak emission wavelength of 585 nm. The amount of fluorescence produced is directly proportional to the amount of D-glucose present in the sample.
The protocol calls for a fluorescence measurement using a 540/35 excitation filter and a 590/20 emission filter.
The experiment data file does not contain any data.
Plate configuration assumes that samples will be run in duplicate. Included is a standard curve of D-glucose concentration (0 to 125 μM) as defined by Invitrogen’s Glucose/glucose oxidase Assay kit. The plate includes 8 standard concentrations, but no blank or controls wells. Thus, 40 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, (3) calculated cholesterol concentrations and statistics of unknown samples.