Immunology: Flow Cytometry
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Flow cytometry is used to...
- - identify, enumerate and isolate cells bearing a particular antigen
- - cells in suspension are labeled by direct or indirect immunofluorescence (addition of fluorescent tag)
Flow cytometry is sometimes also referred to as...
- fluorescent activated cell sorting (FACS)
How a flow cytometer works...
- - 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
Information from flow cytometry is represented in...
- 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
Density dot plots are...
- - 3-dimensional representation of data from 2 different labels
- - x and y axes represent the fluorescent intensity from 2 different labels
A fluorochrome is...
- any molecule or protein that can absorb light at one wavelength (absorption wavelength) then emits light of a lower energy wavelength (emission wavelength)
If using more than one fluorochrome...
- it is best if the absorption wavelengths are the same but the emission wavelengths are sufficiently different so they can be quantitated separately
Two common fluorochromes are...
- fluorescein and phycoerythrin
- Fluorescein (FITC)
- - most commonly used
- - small organic molecule
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