Radboudumc Technology Center Flow cytometry

What is flow cytometry?

Flow cytometry is a sophisticated technique of single cell analysis. It simultaneously measures and analyzes multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light. Some of the characteristics measured include a particle's relative size, granularity or internal complexity and relative fluorescence intensity.

• Technology

  A flow cytometer is composed of three main systems: fluidics, optics and electronics. The fluidics transports the particles in a stream to the laser beam for interrogation. The optics 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 convert the detected light signals into digital signals that can be processed by the computer.
  
  Monoclonal antibodies tagged with a fluorescent dye can be used to identify a particular cell type based on individual surface, cytoplasmic, or nuclear antigens. Different mixtures of fluorochromes can be used to distinguish sub-populations. Dyes, which bind directly to cell components such as DNA and RNA, are used as well.
  
  The flow technology provides a powerful and sensitive tool for investigating the structure and function of cells, bacteria and other particles. These measurements can be made at flow rates of 100-20.000 cells per second.

• Cell sorting

  Cell sorting allows us to capture and collect cells of interest for further biochemical, or functional analysis. Cells can selectively be removed from a mixed population based on any number of physical characteristics, for example, size or fluorescence. A voltage charge is applied to drops containing a cell that meets the predefined sorting criteria. Positively and negatively charged plates deflect the charged drops into collection devices such as tubes or onto microscope slides depending on the charge polarity. Sorted cell preparations can be cultured as sorting can be done under sterile conditions. Additionally when sorting into microplates the system is capable to perform Index Sorting, a valuable tool to establish the correlation between phenotype and gene expressing of sorted cells.
  
  

• Data interpretation

  Some form of graphical representation is a necessary part of the analysis and interpretation of flow cytometric data. This graphical representation permits visualization of the number and inter-relationship of cell populations and is usually a series of graphs and numerical summaries of the results. There are a wide variety of sophisticated computer programs to assist in the display and analysis of flow data.
  

Applications

• Cell phenotyping
• Standardized immune profiling
• Cell sorting based on fluorescent proteins
• 4-way multiple populations sorting
• Multiwell single cell sorting in 6-384 well plates for single analysis applications
• Nucleic acid analysis/Cell cycle analysis
• FRET measurements
• Small particles analysis and sorting
• Cell function analysis (calcium efflux)
• Dynamic multidimensional immune functionality profiling