Flow Cytometry

Flow cytometry is a laser based technology that allows for simultaneous multi-parametric analysis of physical and/or chemical characteristics of cells at the single-cell level. Fluorescence-labeled cells are carried to sensing area in sheath fluid and cross laser beam one a time. Information about the properties of cells, including cell size, granularity by scattered light and relative fluorescence intensity, are transmitted to light detectors after passing through a series of optical filters. This information is then converted into digital electronic signals and collected by specialized computer programs for further analysis.

Flow cytometry technique can be used in a wide range of research applications such as:

• • immunophenotyping,

  • simultaneous analysis of levels of surface and intracellular markers (e.g., intracellular cytokine level),

  • cell viability, apoptosis and proliferation,

  • DNA content and cell cycle analysis,

  • gene expression and transfer (e.g., transfection efficiency),

  • intracellular Calcium flux, and

  • activation states and oxidative burst.

Fluorescence-Activated Cell-Sorting (FACS)

Fluorescence-activated cell sorting is a specialized type of flow cytometry where a heterogeneous mixture of cell suspension can be sorted into up to four populations of interest based on specific light scattering and fluorescent characteristics. Cells are sorted one a time into single-cell-containing droplets that are broken off from the stream by a vibrating mechanism. As droplets flow pass laser beam and light detectors, those with cell of interest that meets the preset criteria are placed with an electrical charge and deflected to different angles before entering designated collection tubes.

FACS can be used for:

• • purification of rare populations such as cancer stem cells,

  • isolation of particular cell sub-populations of interest (e.g., GFP-positive transfected cells, lineage negative bone marrow cells by surface marker staining), and

  • establishment of monoclonal transfected cell line by single cell isolation.