Multicolor Immunophenotyping using Flow Cytometry: Evaluation of Multiple Methods for Instrument Optimization

Abstract

Abstract A basic tool of the Immunologist, multicolor flow cytometry is a technology that uses multiple fluorescent markers to identify and characterize populations of cells. To obtain high quality fluorescent flow cytometry data, a well-optimized instrument is required. The most common type of detector is the photo-multiplier tube (PMT), which amplifies signal from emitted light photons by applying a voltage to the PMT. As the voltage is increased the fluorescent signal is increasingly separated from background, providing greater resolution of the positive signal. However, at a certain voltage, the increasing separation of fluorescent signal from background will plateau and the separation of fluorescent signal from background will remain constant. This is called the minimum voltage required; an ideal minimal voltage will amplify dim signal above background, but is not so high that the fluorescent signal exceeds the upper range of PMT linearity. In most cases, adjusting voltages above the minimal voltage does not increase the separation, while voltages below the minimal voltage requirement compromise detection of dim fluorescent signal. A variety of methods have been proposed to optimize the PMT voltage, most using a technique called the voltage walk, or voltration. This study compares a variety of techniques and calculations, using different types of hard-dyed beads that are detected in all channels but do not include specific fluorophores used for typical experimentation; both cells and antibody-capture beads labeled with fluorophores specific to a detector; Electronic Noise (EN) of the PMT, and methods combining these approaches. For Research Use Only. Not for use in diagnostic procedures.