Flow Cytometry Details

Labeling the Cells of Interest
The sample cells or constituents to be measured are labeled with fluorescent dye molecules conjugated to antibodies that bind specifically to those constituents. There are many stains available. For example, nuclear DNA may be stained with propidium iodide or DAPI, while cell surface antigens may be labeled with monoclonal antibodies conjugated with fluorescent dye such as FITC or phycoerythrin.

Cell Analysis
The suspension of cells or other biological particles is injected into the flow cytometer's nozzle. Constrained to the center of a fluid stream, the cells emerge from the flow cytometer's nozzle and pass single file through an intense beam of excitation (laser) light at the observation point. Each cell or event produces a short flash of fluorescence, the intensity of which is proportional to the cellular content of the fluorescently labeled constituent.

Fluorescence pulses are collected by appropriate optics that focus the light on a sensitive detector, usually a photomultiplier tube (pmt). The detector transforms the pulses of light into electrical pulses that are converted into digital values by the flow cytometer's electronics and then sent to a computer.

Each cell also causes scattering of the excitation light. Scattering is a function of the size, shape, and structure and is recorded for each cell.

Cell Sorting
Additional processes allow the cytometer to separate the desired cells from the sample. The flow cytometer nozzle is vibrated at a high frequency by a piezoelectric transducer, causing the microscopic jet stream exiting the flow chamber to break into discrete droplets. As a cell of interest reaches the breakoff point, positive or negative charging is applied to the cell.

The charged droplets pass through a pair of vertical deflection plates, one charged at a negative voltage and the other at a positive voltage. The positively charged droplets shift toward the negative plate and the negatively charged droplets shift toward the positive plate. The droplets then fall into sample receptacles below. Uncharged droplets continue in a straight line out of the flow chamber to an aspirated waste collection tube below.

This charging of the droplets allows sorting of particles with two attributes per run by positive or negative charging. The MoFlo cytometer can sort four samples of interest per run by charging the events with a range of electrical charges.