Flow Cytometry

Flow Cytometry

Flow cytometry is a means of identifying and measuring certain physical and chemical characteristics of cells or particles as they travel in suspension one by one past a sensing point. The benefit of flow cytometry is the rapid simultaneous measurement of several parameters on a cell by cell basis. This is in contrast to the more traditional biochemical measurement of individual parameters to give a final mean value per cell. A major benefit of this approach has been to address the question of accurate enumeration of minor cell populations, which have always been problematic to estimate by manual microscopic methods. In Flow Cytometry we measure light, freuently fluorescent light. If you are not familiar with issues concerning fluorescence and how those relate to biological work, there are some good tutorials on the topic here and here.

There are a number of applications for flow cytometry, including, but not limited to:

Immunophenotyping - Using fluorescence-conjugated antibodies directed toward a protein(s) of interest, cells expressing that protein(s) on the surface or intracellularly may be detected by flow cytometry. Specific cell types may be distinguished within a mixed population using multiple fluorescence-conjugated antibodies.

Transfection efficiency may be determined when a fluorescent protein (i.e. GFP) is used as a marker.

Apoptosis measurement - Several flow cytometric methods to detect apoptosis are available.

Cell cycle analysis - Fixed cells are stained with a dye that binds to DNA (e.g. propidium iodide). Fluorescent intensity is used to determine the amount of cellular DNA present in each cell (i.e. two copies of a genome have roughly twice the fluorescent intensity of one copy).

Cell proliferation - Carboxyfluorescein diacetate, succinimidyl ester (CFSE) is a dye that diffuses into cells and is passed from parent to daughter cells so that the cells of each generation have half the fluorescent intensity of their parent cells.

Cell sorting - A particular subset(s) of cells may be sorted from a mixture of cells based upon particular properties. Currently we have a Miltenyi magnetic sorter, with a Flow sorter joining the line up soon.

Membrane potential - Bacterial membrane potential may be analyzed using DiOC2, which exhibits green fluorescence in all bacterial cells, but shifts to red fluorescence as the dye becomes more concentrated in cells with larger membrane potentials. Mitochondrial membrane potential may be analyzed in the same manner with JC-1.

Cytometric Bead Arrays - CBAs allow the detection of specific soluble proteins from serum, cell culture supernatant, or cell lysates. The CBA Flex Sets provide the flexibility of choosing from 1 to 72 different soluble proteins for analysis from a wider range of species.

Live/dead bacteria discrimination - You can test how fast an antibiotic is killing microbes: live cells have intact membranes and are impermeable to dyes such as propidium iodide, which only leaks into cells with compromised membranes. Thiazole orange enters all cells, live and dead, to varying degrees. Thus a combination of these two dyes provides a rapid and reliable method for discriminating live and dead bacteria.

There is an abundance of Flow Cytometry literature. One particularly useful about interpreting Flow data: interpretingflow.pdf.

Right now the limits of the technology stand around detecting 30 parameters at the same time. Here is a paper describing what sort of things to think of when running a complex experiment- although even if you are planning on using only a few colors it is worth reading it. 17colorflow.pdf.