Immunology: Flow Cytometry

• - identify, enumerate and isolate cells bearing a particular antigen
•  - cells in suspension are labeled by direct or indirect immunofluorescence (addition of fluorescent tag)

- fluorescent activated cell sorting (FACS)

• - cells exit a flow cell and are illuminated with a laser beam
• - the amount of laser that is scattered off the cells as they pass through the laser can be measured and gives information concerning the size of the cell
• - the laser can also excite the fluorochrome on the cells and the fluorescent light emitted by the cells can be measured by one or more detectors

- histograms & dot plots

- one parameter shows increasing amounts of fluorescence plotted on x axis and the number of cells exhibiting the amount of fluorescence is plotted on the y axis

• - 3-dimensional representation of data from 2 different labels
• - x and y axes represent the fluorescent intensity from 2 different labels

- any molecule or protein that can absorb light at one wavelength (absorption wavelength) then emits light of a lower energy wavelength (emission wavelength)

- it is best if the absorption wavelengths are the same but the emission wavelengths are sufficiently different so they can be quantitated separately

- fluorescein and phycoerythrin

• - most commonly used
• - small organic molecule