What is Flow Cytometry?

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COMPILED AND CIRCULATED BY DR. PRITHWI GHOSH, ASSISTANT PROFESSOR, DEPARTMENT OF BOTANY, NARAJOLE RAJ COLLEGE

BOTANY: SEM-VI, PAPER-DSE4T: ANALYTICAL TECHNIQUES IN PLANT SCIENCES, UNIT-1: FLOW CYTOMETRY

What is Flow Cytometry?

‘Flow Cytometry’ as the name suggests is a technique for cell counting and measurement of different properties of the cell (‘cyto’= cell; ‘metry’=count/measurement). It is a laser-based technology that measures and analyses different physical and chemical properties of the cells/particles flowing in a stream of fluid through a beam of light.

Components of a Flow Cytometer: A flow cytometer is made up of three main systems: fluidics,

optics, and electronics:

• The Flow system (fluidics) system transports particles in a stream to the laser beam for interrogation.

• The Optics system (light sensing) 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 (signal processing) 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.

Basic mechanism

• Biological sample

• Label it with a fluorescent marker

• Cells move in a linear stream through a focused light source (laser beam)

• Fluorescent molecule gets activated and emits light that is filtered and detected by sensitive light detectors (usually a photomultiplier tube)

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• Conversion of analog fluorescent signals to digital signals

Working of a Flow Cytometer

In the flow cytometer, particles are carried to the laser intercept in a fluid stream. Any suspended particle or cell from 0.2–150 micrometers in size is suitable for analysis.

• The portion of the fluid stream where particles are located is called the sample core.

• When particles pass through the laser intercept, they scatter laser light. Any fluorescent molecules present on the particle fluoresce.

• The scattered and fluorescent light is collected by appropriately positioned lenses.

• A combination of beam splitters and filters steers the scattered and fluorescent light to the appropriate detectors.

• The detectors produce electronic signals proportional to the optical signals striking them.

Potential applications for flow cytometry

The potential applications of flow cytometry analysis are numerous, including the detection and measurement of:

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• Protein expression—throughout the entire cell, even the nucleus

• Protein post translational modifications—includes cleaved and phosphorylated proteins

• RNA— miRNA, and mRNA transcripts

• Cell health status—from viability to late-stage apoptosis or programmed 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 even regulatory T cells

An additional capability of specialized flow cytometers is the ability to sort cells and recover the subsets for post experimental use. This specialized flow cytometer is called a fluorescence activated cell sorter (FACS), a term that is sometimes erroneously used interchangeably with ‘flow cytometer’. This usage is incorrect. A flow cytometer is an analytical machine that does not perform cell sorting. Cell sorters use fluidics and fluorescence components similar to those in flow cytometers but are able to divert a specific population from within a heterogeneous sample into a separate tube, typically based on specified fluorescence characteristics. If collected under sterile conditions, these cells can be further cultured, manipulated, and studied.

References

1. https://www.abcam.com/protocols/introduction-to-flow-cytometry

2. http://www.biologydiscussion.com/biochemistry/flowcytometry/principles-of- flowcytometry-with-diagram/12989