Flow Cytometry (FCM) /FACS Introduction

Flow cytometry is a widely used method for analyzing the expression of cell surface and intracellular molecules, characterizing and defining different cell types in a heterogeneous cell population, assessing the purity of isolated subpopulations and analyzing cell size and volume. It allows simultaneous multi-parameter analysis of single cells.

It is predominantly used to measure fluorescence intensity produced by fluorescent-labeled antibodies detecting proteins, or ligands that bind to specific cell-associated molecules such as propidium iodide binding to DNA.

The staining procedure involves making a single-cell suspension from cell culture or tissue samples. The cells are then incubated in tubes or microtiter plates with unlabeled or fluorochrome-labeled antibodies and analyzed on the flow cytometer.

Principle of Flow Cytometer

A typical flow cytometer should include three main sub-systems: the fluidics, the optics and the electronics. Cells in suspension flow in single‐file through an illuminated volume where they scatter light and emit fluorescence. Light and fluorescence is collected, filtered and converted in optical bench. Light were converted to digital signals that are analyzed and stored on a computer.

One of the fundamentals of flow cytometry is the ability to measure the properties of individual particles. When a sample in solution is injected into a flow cytometer, the particles are randomly distributed in three-dimensional space. The sample must therefore be ordered into a stream of single particles that can be interrogated by the machine's detection system. This process is managed by the fluidics system.

Cartoon of Flow Cytometry (FCM) / FACS Fluidics

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See "What is Flow Cytometry" for details