Question

Describe the following terms in reference to flow cytometry analysis of immune cells: hydrodynamic focusing, forward light scatter, side light scatter, 2 dimensional scatter plot, threshold, bandpass filter, fluorophore, and CD marker?

Answer 1

Hydrodynamic Focusing in flow cytometry:
Hydrodynamic focusing is a technique that enables users of flow cytometry cells to gauge the size of particles in a flow channel, whether they be blood cells, viruses or bacteria. As particles enter the chamber they will typically pass through a laser beam which causes a temporary disruption in the optics.

Forward light scatter in flow cytometry:
One detector measures scatter along the path of the laser. This parameter is referred to as forward scatter (FSC). The measurement of forward scatter allows for the discrimination of cells by size. FSC intensity is proportional to the diameter of the cell, and is primarily due to light diffraction around the cell.

Side light scatter in flow cytometry:
Side light scatter is a measurement of mostly refracted and reflected light that occurs at any interface within the cell where there is a change in refractive index.

2 dimensional scatter plot in flow cytometry:
A two dimensional scatter-plot compare 2 parameters simultaneously in a flow cytometry data. Two axes represent two detectors and cells are presented by dots.

Threshold in flow cytometry:
A threshold is the lowest signal intensity value an event can have for it to be recorded by the cytometer. The trigger channel is the critical parameter used to determine if an event should be recorded. When using the BD Accuri C6 flow cytometer, setting the primary threshold also defines the trigger channel.

Bandpass filter in flow cytometry:
Bandpass filters are the ones that are most commonly used in flow cytometry. Bandpass filters are named based on the center and width of the band of light that will pass through.

Fluorophore in flow cytometry:
Fluorophores are fluorescent markers used to detect the expression of cellular molecules such as proteins or nucleic acids. Before considering the different types of fluorophores available for flow cytometry, it is necessary to understand the principles of light absorbance and emission.

CD marker in flow cytometry:
CD Markers are especially useful for identification of leukocyte population using flow cytometry. The great advantage of flow cytometry is that it allows for the simultaneous detection of several markers on a single cells at the very same time.