Flow cytometry

Principle and purpose

Flow cytometry makes it possible to measure and analyze simultaneously and on a large number of particles (cells, bacteria, beads, etc.), several constituents of the same particle defined by several parameters: the size (FSC, Forward Scatter), the granulosity or texture (SSC, Side Scatter), and fluorescence (FL).

This technology consists in scrolling, in a liquid vein, the cells in front of a laser beam. The cells thus aligned one behind the other, are analyzed thanks to the coupling of an optical system to electronics (photomultipliers), which records the parameters of light diffusions and fluorescences.
The main advantages of this technology are:

  • multiparametric analysis (size, granulosity and fluorescence) and simultaneous analysis on a very large number of cells, and the speed of data acquisition;
  • the possibility of analyzing cell subpopulations, even poorly represented, in a mixture of several types of cells having at least one different parameter.

Flow cytometry therefore makes it possible to carry out experiments on cells with specific fluorescent probes, in addition to or instead of confocal microscopy analyzes.



Equipment


The MACSQuant Analyzer: compact and space-saving (benchtop cytometer), quiet and relatively easy to use

The INCI has a flow cytometer MACSQuant Analyzer type (Miltenyi Biotec). The properties and characteristics of this device are:

  • 3 lasers (violet 405 nm, blue 488 nm and red 635 nm) allowing the analysis of 9 optical parameters (FSC, SSC and 7 fluorescence channels FL1 to FL7);
  • Automatic calibration of voltages;
  • Multiple assay configuration: 5 ml, 15 ml, 50 ml tube rack, 96 well plate rack and single tube sample holder;
  • Fast analysis: up to 10,000 events / sec;
  • Absolute count of cells (volumetric);
  • Pre- and post-acquisition compensation possible (software with an automatic compensation matrix);
  • Automatic maintenance: wash cycles at start-up, shutdown and between 2 acquisitions; washing / decontamination system with programmable standby;
  • Analysis software running Windows XP. Export of datas possible in FlowJo.


Applications

  • Cell count, proliferation and cell cycle analysis;
  • Cell viability and apoptosis;
  • Cell transfection analysis, followed by stable lines;
  • Immunophenotyping;
  • Analysis of phagocytosis in macrophages;
  • Membrane surface markings and protein internalization;
  • Exocytosis and endocytosis analysis (measurement of secretion).


Contact

The INCI Flow Cytometry Facility is located on the 2nd floor of the Neurochemistry Center (2007). Its mission is to provide researchers and INCI staff with flow cytometry technology.

Scientifique support: Stéphane Ory, CR1 CNRS