Analysis

 

Flow cytometry is a powerful and effective tool for analyzing the structural and functional characteristics of cells or particles in suspension. A test tube containing a single cell suspension is placed on the instrument where the sample is pushed by air under pressure through tubing where it is injected into a nozzle. The nozzle, containing sheath fluid under pressure, hydrodynamically focuses the cells so that they exit the nozzle in single file down the center of the stream resulting in laminar flow. The flow rate can be controlled by adjusting the differential pressure between the sheath fluid and the sample injection pressure. Laser light is then focused onto the stream where each cell is excited as it passes through the laser beam.

Photo multiplier tubes (PMT's) are detectors which collect the photon emissions from each "event" and convert them to analog voltages. The analog signals are then digitized by analog to digital converters (ADC's) and recorded as data files. Optical filters are placed before the detectors so that only wavelengths of light corresponding to specific fluorochrome emissions are collected by each detector (e.g. FITC emits in the green region therefore a 30 nm bandpass filter centered at 520 nm could be used to collect light from this fluorochrome). Light scattered at the same wavelength and direction as the laser light, primarily from the surface of the cell, correlates with relative cell size (Forward Angle Light Scatter) while light scattered 90 degrees to the laser (Side Scatter) usually from internal structures, correlates with granularity. By correlating these two parameters, one can discriminate subpopulations of cells in peripheral blood samples, for example. Signals corresponding to cell debris or cell aggregates can also be detected and excluded from analysis on the basis of forward and side scatter.

Staining cells with multiple fluorochromes conjugated to antibodies or fluorochromes directed at other specific targets such as DNA, cytokines, or other proteins distinguishes cell subpopulations which can be quantified. Data is displayed and analyzed using histograms or two-dimensional dot plots on a computer system.

The Cytometry Core Facility has two Analyzers available for use: a single-laser BD FACSCanto II and a five laser BD LSR II.