Method of the Month: Flow Cytometry
October 2023
Background
How can we determine the exact number of cells in a tissue sample? In medical laboratories that process blood products and tissue samples, technicians use a procedure called flow cytometry to accurately count cells and gather information about protein expression. Developed in 1968 by Wolfgang Göhde, and later commercialized by manufacturers, flow cytometry is a powerful tool that can be used in numerous fields of biology.
Method
Flow cytometry is a lab technique that measures a variety of cell properties carried out in a machine called a flow cytometer. Flow cytometers consist of three main systems: the hydrodynamic focusing system, the detection system, and the conversion system.
The hydrodynamic focusing system uses liquid to force a sample of cells to travel at a constant speed through the flow cytometer. A prepared sample is poured into the main stream. The stream is created by sheath fluid, a saline solution. The sheath fluid runs down the funnel in a streamlined focus through the hole at the bottom, pushing the sample along with it. The hole in the funnel must be small enough that only one cell can drop at a time. At the bottom of the flow cytometer, there is a waste container to collect the fallen liquid material.
The detection system is made up of lasers and mirrors. As the cells drop one by one from the hydrodynamic focusing system, a laser, made up of multiple colored lasers, is shot at the falling cells. The cells scatter (refract) the light in many different directions. The forward scatters of light are caught by the primary detector and are refracted in the same line, parallel to the laser. The side scatters of light are caught by a detector perpendicular to the laser and slightly angled upwards. A final detector assesses the fluorescence intensity antigen density (FIAD) of side-scattered light. This detector measures the excitement of the fluorochrome (fluorescent chemical dye attached to antibodies), capturing the intensity and color of the light that is reflected off of the cell. Each of these detectors sends information to the conversion system.
The conversion system is comprised of computers that interpret the data gathered by the detection system. Each detector and type of scattered light tells a different story about the cell. The forward scattered light shows size and morphology (shape); the side scatter shows the complexity/granularity (being composed of several elements) of the cell; the scatter caught by the fluorescence detector shows the expression of a certain protein. All of the information gathered is put into graphs that allow scientists to interpret what type of cell has passed through the laser, as well as the ratio of each type of cell in a certain sample.
Application
Let’s examine a specific application of this procedure: consider a sample of T cells that have been genetically engineered to make a protein called antiCD19. To prepare this sample, scientists add antibodies, which attach to the antiCD19 proteins made by the cells. They then add fluorochrome that binds to the antibodies and reflects green light. They run this prepared sample through a flow cytometer. Each cell passes individually through the hydrodynamic focusing system, gets interrupted by the laser, and scatters light to the different detectors. The conversion system then categorizes the types of cells that pass through the laser, displaying the results on a graph. The data collected also includes the ratio of cells, the proportion of the T-cell sample, and the percent transduction (the percentage of T-cells that reflected the green light). The cells that reflected the green light were those that expressed the antiCD19 protein. This type of information has many applications in medicine and research, making flow cytometry a vital component of a molecular biologist’s toolkit.
For more information on flow cytometry, check out the following links!
vhttps://www.beckman.com/resources/technologies/flow-cytometry/history
