Cytometry solutions

Flow cytometry is a laser-based technology that is used in the detection and measurement of physical and chemical characteristics of cells or particles as they flow in a fluid suspension across an illuminated light path. 

Flow cytometry is a powerful technique that is widely used to identify and characterize different immune cells in heterogeneous samples. It primarily relies on the use of fluorescently-labeled molecules to identify and characterize antigen profiles in a variety of research applications and for clinical diagnostics on a single-cell basis.

By using different fluorochromes with emission wavelengths that span across the color spectrum, proteins associated with surface, cytosolic, and nuclear compartments can be detected simultaneously.

Flow cytometry is an incredibly versatile technique and many variants of the technique have been developed in recent years. Fluorescence-activated cell sorting (FACS) is a derivative of flow cytometry that adds an enhanced degree of functionality. In practice, the terms “flow cytometry” and “FACS” are often used interchangeably

Flow cytometry has proven to be an invaluable analytical tool in the field of disease diagnosis. Vast numbers of cells can be analyzed in a matter of minutes, conferring advantages for study over other analysis methods such as Cellometer vision or microscopy.

Cytometry applications for analysis

In a Flow Cytometer:

• Sample cells are passed through a narrow channel one at a time.
• Light is used to illuminate the cells in the channel.
• A series of sensors detect the types of light that are refracted or emitted from the cells.
• Data acquired by the sensors is compiled and integrated to build a comprehensive picture of the sample.

Flow cytometry uses fluorescent markers to identify and analyze cell components.  DNA, RNA, proteins, or other molecules in the cell are labeled with fluorescent dyes. If laser pulses are then “fired” at the cell, the labeled molecules are illuminated briefly. 

Light emitted in each cell can then be detected by the flow cytometer and the relative amount of each component can be measured due to its intensity. This is a crucial procedure for disease diagnosis and prognosis.

Flow cytometry data analysis is built upon the principle of gating. Gates and regions are placed around populations of cells with common characteristics, usually forward scatter, side scatter and marker expression, to investigate and to quantify the populations of interest. 

Gating allows a researcher to gather and display more information about a subpopulation of cells than could normally be displayed on a 2- or 3-dimensional dot-plot.

A flow cytometer is made up of three main systems: fluidics, optics, and electronics. 

• The fluidics system transports particles in a stream to the laser beam for interrogation.
• The optics system consists of lasers to illuminate the particles in the sample stream and optical filters to direct the resulting light signals to the appropriate detectors.
• The electronics system converts the detected light signals into electronic signals that can be processed by the computer. For some instruments equipped with a sorting feature, the electronics system is also capable of initiating sorting decisions to charge and deflect particles

A flow cytometry machine is used to perform the following tasks that are important for disease diagnosis:

• Blood cell counts
• Observing different types of leukocytes in a sample
• Sorting T cells to determine how their function has been affected by an infection
• Determining the total DNA content in cells when performing tumor biopsies in cancer research
• Studying the effect of infectious diseases on individual cells with cell sorting
• Detecting pathogenic microbes in both biological and environmental samples
• Detecting minimal residual disease cells in bodily fluids

Although flow cytometry is well established as a cell biology technique, the development of high-throughput flow cytometry (HTFC), enabled by the introduction of faster plate-based sampling, has transformed the technology into an attractive drug discovery platform as well.

A few of the major applications flow cytometry is used for (in the scope of modern clinical settings) include: 

• Protein expression—throughout the entire cell, even the nucleus
• Protein post translational modifications—includes cleaved and phosphorylated proteins
• RNA—including both miRNA, and mRNA transcripts
• Cell health status—detection of apoptotic cells or cell death
• Cell cycle status—providing a powerful tool to assess cells in G0/G1 phase versus S phase, G2, or polyploidy, including analysis of cell proliferation and activation
• Identification and characterization of distinct subsets of cells within a heterogeneous sample—including distinguishing central effector memory cells from exhausted T cells or regulatory T cells

Cell selection

Compared to other isolation techniques like magnetic bead separation and panning, flow cytometry offers these advantages:

• Higher purity 
• Can separate cell types based on their level of fluorescence intensity
• Better separation of populations using multiple antibodies
• Higher recovery of cells of interest
• Can eliminate dead cells
• Can sort based on internal staining (DNA, cytokine expression, GFP, etc.)
• Can sort up to four populations simultaneously

Disadvantages, or limitations to flow cytometry include:

• The laser can only analyze one cell at a time 
• Cells must be in suspension to be analyzed (thereby restricting the analysis of tissue) 
• Highly trained operators are required
• Cells must be viable to be analyzed

Explore our cytometry capabilities

Flow cytometry has evolved from a promising new technology to an indispensable scientific diagnostic tool. And the frontier of flow cytometry applications continues to expand. 

Avantor has the knowledge, products and services to help enable the advancement of flow cytometry and science throughout the world. We do this by encouraging innovation and striving for process excellence. 

At Avantor, we supply what is needed to bring science to life. By providing superior product and service solutions, we catalyze innovation and empower the success of our customers.