The principle of flow cytometry is the segregation and analysis of cells which are arranged in a single line as they flow past a detector in a stream.
A machine sorts and counts the cells that have been labelled or tagged using fluorescent dyes or other methods.
The labels that are used depend on the lamp or laser that is used in the analyzer. This lamp or light excites the fluorochromes which are detected by the device.
There are several labels or fluorophores that are used in flow cytometry. These fluorophores or fluors are generally attached to an antibody that recognises a target feature either displayed on the cell or inside it.
Fluors can also be attached to a chemical that is attracted to a structure of the cell such as its membrane. Each of the fluors has a particular peak excitation and emission wavelength.
The types of labels used depends on the wavelength of the laser that excites the fluors as well as on the available detectors.
Sometimes, quantum dots are used instead of fluors because the dots have such narrow emission peaks.
In mass flow cytometery, multiple labelling is performed using lanthanide isotopes to antibodies. This allows for as many as 30 labels to be used.
Examples of some of the labels available are given below:
- Blue argon laser (488 nm) – This is an air-cooled laser and is therefore easier to set up and use. It is used in single laser machines. Some of the labels used for this laser include:
- For Green channel on FL1 - Alexa Fluor 488, GFP, FITC, CFSE, DyLight 488 or CFDA-SE is used
- For Orange channel on FL2 – PE or PI is used
- For Red channel on FL3, usually - PE-Alexa Fluor 700, PerCP-Cy5.5, PerCP, PE-Cy5.5 or PE-Cy5 is used
- For Infra-red channel usually on FL4 (this feature is not available for all flow cytometers) - PE-Cy7 or PE-Alexa Fluor 750 is used
- Red diode laser (635 nm) – The labels used include APC-Cy7, APC-eFluor 780, Alexa Fluor 700, Cy5, APC, Draq-5
- Violet laser (405 nm) – The labels used include Amine Aqua, Pacific Orange, Pacific Blue, DAPI, Alexa Fluor 405, eFluor 450, eFluor 605 Nanocrystals, eFluor 625 Nanocrystals or eFluor 650 Nanocrystals
Reviewed by Sally Robertson, BSc