Bead Based Assays

Tione Buranda (UNM School of Medicine)

 

Instrument: Becton Dickenson Flow Cytometer and Spectrofluorometer

 

This lab will focus on the spectroscopic concepts and application of fluorescence calibration beads as an approach to quantify measured fluorescence intensities associated with cells or particles. These measurements are important for intra- and inter lab comparisons of data when different instruments, reagents and labs are involved. The intensity of emission associated with cells or particles and measured at the flow cytometer is directly proportional to, I0ef%T;  where I0 is the intensity of an attenuated light source that does not saturate the fluorophore, e is the absorption coefficient of the fluorophore, f is the quantum yield of the fluorophore and %T is the percent fraction of emitted light that is transmitted by the bandpass filter at the detector PMT. This lab will examine the methods by which the molecular parameters (ef %T) are measured for various samples and used together with calibration beads to quantify flow cytometry measurements. With this in mind students will learn standardization of instruments, reagents and assays essential for quantitative flow cytometry.