Flow Cytometry Guide: Glossary

Author: Mike Blundell | Reviewer: Chloe Fenton

Apoptosis – Programmed cell death through various tightly regulated biological pathways.

Antibody – A specialized protein of the immune system that can identify and neutralize specific targets called antigens.

Area – The integral of the pulse.

Autofluorescence – Natural levels of fluorescence found within cells due to the presence of fluorescent compounds.

Backgating – A useful gating control to ensure you have identified the right cell population using traditional gating.

Band pass filter - Allow light through within a specified narrow range.

Cell sorting – The ability to separate cells, identified by specified characteristics, within droplets using an electrical charge.

Compensation – Mathematical algorithm for removing fluorescence spillover of one fluorophore into multiple detectors.

Doublets – Where two particles pass through the laser at the interrogation point.

Drop delay – The time between the interrogation and the point where a droplet breaks off during cell sorting.

Event – Any particle which generates enough signal when it passes through the laser to be recorded as a signal or pulse.

Fc receptors – Antibody receptors on certain cells which bind antibodies via their constant region to elicit immune responses.

Fixation – Crosslinking of cellular proteins to preserve from decay and allow permeabilization without loss of cell contents and structure for intracellular staining.

Forward scatter – Light that is scattered in the forward direction (up to 20^o) after interacting with a particle.

Flow cytometer – Instrument which allows the measurement of properties of individual particles as they pass through a laser.

Fluorescence minus one control – Specific control, where one fluorophore is removed from the staining panel, to account for fluorescence spread.

Fluorescent protein – A protein which can accept light energy and re-emit at longer wavelengths and can be expressed in cells for live marking.

Fluorophore – Fluorescent markers that accept light energy at a given wavelength and re-emit at a longer wavelength.

Gating – Placing of regions around populations of cells with common characteristics to quantify and further investigate.

Height – The maximum amount of current output by the PMT of the pulse.

Histograms – Plots which display a single measurement parameter.

Hydrodynamic focusing – A faster outer sheath fluid around the sample stream allows narrowing of the stream creating a stream of single cells.

Immunophenotyping – Identification of cells in a population through the staining and identification of specific markers.

Instrument configuration – The set-up of lasers, optics and filters contained within the cytometer.

Isotype controls – Antibodies raised against an antigen not found on the cell being analyzed to help determine specific antibody binding.

Laser – A device that emits optically amplified light at a single wavelength.

Long pass filter – Allow light through above a certain wavelength.

Maximal emission – The wavelength at which a fluorophore emits the most photons.

Maximal excitation – The wavelength at which a fluorophore is excited by the most photons.

Parameter – The measurement from any given detector which can be displayed in height, area or width.

Photo multiplier tube - A photoemissive detection device in which the absorption of a photon results in the emission of an electron.

Permeabilization – Creation of holes in the cell membrane using detergents to allow large molecules (such as antibodies) to enter the cell for intracellular staining.

Resolution – The ability to separate a positive from a negative population.

Short pass filter - Allow light through below a certain wavelength.

Side scatter – Light that is scattered at 90^o after interacting with a particle.

Spillover – The overlap of one fluorophore emission spectra with another.

Spread – The increase in a population’s fluorescence into another channel after compensation.

Stain index – The point where there is maximal separation between the positive and negative population on stained samples.

Stokes Shift – The difference between the maximal excitation and emission wavelengths of a fluorescent molecule.

Tandem dye – A fluorescent dye which is two molecules covalently coupled together.

Threshold – Signal intensity below which the flow cytometer does not record an event.

Titration – Dilution of an antibody to the concentration at which there is an optimal stain index.

Viability dye – Dye which allows identification of dead cells through a reduction in cell membrane integrity.

Width – The time interval during which the pulse occurs.