Why use flow cytometry (FCM)? FCM is FAST, analyzing 500 to 5000 suspended particles (usually cells) per second. FCM is MULTIPARAMETRIC, allowing for the measurement of up to six structural and functional phenomenon at the same time. FCM is not a bulk assay; measurements are made on INDIVIDUAL CELLS, one at a time. Finally, FCM allows the SORTING of heterogeneous populations.


How does FCM work? There are three main systems in a flow cytometer: fluidic, optical, and electronic. The fluidics system pumps a suspension of cells through a nozzle, creating a stream with laminar flow properties. The optical system focuses one or more laser beams on the stream and collects scattered and fluoresced light from cells as they pass through the laser beam. The electronics measure and digitize these light emissions so that they can be analyzed on a desktop computer. The electronics also control the sorting hardware, based on the light emission characteristics of each cells that passes through the cytometer.


What kinds of parameters can be measured by FCM?

SCATTER PARAMETERS

Forward Scatter (FSC, FS, FALS, etc.) This parameter is a rough indicator of a cell's size.

Side Scatter (SSC, SS, RT. ANG., 90, etc.) This parameter is a rough indicator of cellular granularity, membrane complexity, number of organelles, etc.

Taken together, the two scatter parameters give a morphological fingerprint of whatever is flowing through the cytometer. In addition to the intact, non-aggregated cells that are present in cell suspensions, there are a variety of other particles, including cells debris (smaller) and clumps (larger). Although it is possible to "gate" flow cytometry data based on scatter parameters, this is no substitute for preparative steps that minimize fragmentation and clumping.

FLUORESCENCE PARAMETERS

Immunofluorescence: proteins are detected with antibodies that have been conjugated to fluorescent molecules such as FITC, PE, Texas Red, APC, etc. Detection of molecules on the cell surface (immunophenotyping) is most common, but with a few modifications, proteins can be identified in the cytoplasm (e.g. cytokines), or in the nucleus (e.g. cell cycle control proteins).

Cell cycle/proliferation: DNA content can be measured with PI, 7-AAD, the Hoechst dyes, or DAPI. BrdU incorporation for cell proliferation can be measured by immunofluorescence. This is often done along with DNA staining.

Apoptosis can be measured in a number of ways. The TUNEL technique identifies DNA strand breaks. Annexin V labeling detects changes in the plasma membrane asymmetry. Nuclear condensation and DNA loss are demonstrated by hypodiploid peaks in DNA content experiments. Uptake of Hoechst 33258 has also been shown to increase with apoptotic changes.

Mitochondrial function (Rh123, DiOC6)

Oxidative burst (HE, DCFH-DA, DHR)

Reporter genes (GFP, LacZ, along with the substrate FDG)

Glutathione/reductive reserve (MCB)

Ca++ flux (Indo-1)

Enzyme activity (fluorescent substrates)

pH (SNARF)

Tracking of cell division and conjugation (fluoroscein, calcein, PKH26)

Total protein content (SR101, fluoroscein)

Cell-mediated cytotoxicity