A flow cytometer consists of several component systems; fluidics pumps, lasers, fluorescence detectors, signal converters and computers. When combined, these systems can be used to analyze the physical characteristics of particles or cells in suspension and sort them from mixed populations.
When samples are placed on a flow cytometer, particles are taken up in saline which carries them into a flow cell where they pass by a laser one at a time in single file fashion. As the cells move by the laser, intrinsic and extrinsic properties can be measured and then translated for interpretation on a computer display.
Measurable intrinsic cellular properties include relative cell size and complexity. These characteristics are based on the light scattering properties of cellular membranes both inside and outside the cell and provide the basis for selecting a population of cells from a non-uniform mixture for further analysis or isolation.
Determinations of other cellular properties are based on measures of fluorescence. Molecules on the cell can be specifically labeled with antibodies which are tagged with various light emitting molecules (fluorochromes). The presences of these labels are revealed after their tags are excited by laser light. These tags emit light (fluoresce) at wavelengths longer than the incident laser light and are detected by photomultiplier tubes (PMT’s) set up to collect emissions at specified wavelengths. Tagging cells with antibodies bound to multiple fluorophores can give information about many different cellular proteins both inside and outside of the cell at once. In some cases up to 18 different colours can be measured simultaneously depending on the instrument used. In the end, the relative intensity of the fluorescent emissions can be related to cellular responses after known treatments or used to assess the signs of disease progression in complex immunological disorders such as HIV and autoimmunity.
Flow Cytomety Educational Resources:
DeNovo Software (Michael G. Ormerod): http://flowbook.denovosoftware.com/ (a brand new easy to read text book about flow cytometry)BD Biosciences: ttp://www.bdbiosciences.com/immunocytometry_systems/support/training/online/ITF/index.shtml
Molecular Probes: http://probes.invitrogen.com/resources/education/
Wikipedia.org http://en.wikipedia.org/wiki/Flow_cytometry
Purdue University: http://www.cyto.purdue.edu/flowcyt/educate/pptslide.htm
Science Park: http://sciencepark.mdanderson.org/fcores/flow/files/Operation.html
OCIR Workshop on Flow Cytomety in Cancer Stem Cell Research (Cynthia Guidos)
New Approaches to Multicolor Compensation and Data Visualization (Cynthia Guidos)
Fluorescence Compensation: How to set up your experiment (Jennifer Wilshire)
What is Polychromatic Flow Cytometry? (Mark Kukuruga)
Multicolour Panel Optimization (Holden Maecker)