Citofluorimetry

Alberto Azzalin
0382 98644  alberto.azzalin@unipv.it

Flow cytometry is a methodology that allows both qualitatively and quantitatively measurements of heterogeneous cell suspensions or particles. Nowadays technology allows the simultaneous analysis of multiple parameters both physical (cell size and complexity) and biochemical/molecular (depending on the cellular target analyzed). The underlying principle is based on the use of a light source that emits at variable wavelengths hitting each single cell that flows linearly: deviated-rays (“scatter”) provide the physical information, fluorescence emission provides the information related to the highlighted cellular target (typically with a fluorophore-labelled  antibody). Two types of “scatter” are measured: “forward scatter” (FSC), which gives information on cell size, and “side scatter” (SSC) which gives information on cytosolic granularity.

There are two types of flow cytometry: a classic “non-sorting” type that collects only “scatter” data and fluorescence emissions, and a “sorting” type that uses this information to separate individual cell sub-populations with specific parameters, from initial heterogeneous population.

Versatility and resolutive capacity of flow cytometry allow its diffusion in different areas, from biology to medicine, through pharmacology. In life science, applications are based on the cellular marker analyzed (membrane, cytosol content, organelles, nucleus): cell cycle analysis, blood cells characterization, cell death analysis etc.

Instruments

Cytometer BD FACS Lyric (Becton Dickinson) The BD FACS Lyric flow cytometer is a combination of all cytometers features like simplicity, speed and automation to ease workflow, a standard for clinical cell analysis but also suitable for research activity. Our instrument is equipped with 3 excitation lasers (405, 488 and 642 nm), it acquires up to 12 fluorescence channels, at the maximum acquisition rate of 35.000 events per second with no limit on the number of events acquired. The sensitivity of the BD FACS Lyric is 85 MESF for FITC and 20 MESF for PE. Our system is additionally equipped with 30 or 40 tube autoloader and the fluidics design enables a large selection of sample input devices. Data can be analyzed at the facility using the “BD FACS Suite” software (v1.3) or exported to FCS.

Cytometer ImageStreamX MARK II (Amnis/Luminex) ImageStreamX MarkII combines features of standard flow cytometry and microscopy, performing fast and quantitative analysis of cells thanks to the sensitivity of a cytometry and the spatial resolution of a microscope (at 60x a pixel size is 0.1×0.1 mm). Our system is equipped with 488 nm and 642 nm excitation lasers, one TDI CCD camera to acquire up to 6 images of each cell in flow, up to 5000 cells per second (brightfield, darkfield and 4 fluorescence images) and three objectives for 20x(0.5NA), 40x(0.75NA) and 60x(0.9NA) magnification.

The “Inspire” software (v.200.1) allows the instrument to acquire brightfield and fluorescent images of cells at high speed, while the “IDEAS” software (v.6.2) perform quantitative analysis of cellular images and population statistics. The Instrument allows to analyze several biological processes like translocation, co-localization, cell cycle, apoptosis, autophagy, vesicles internalization and other cellular features.

Cell sorter BD FACS ARIA III (Becton Dickinson) The BD FACS Aria III sorter is based on patented technologies and allow cell sorting to a wide range of applications in research. Its fluidics and optical systems include innovations like the laser excitation optics, the patented flow cell with gel-coupled cuvette and the patented octagon and trigon modules. The sensitivity of the BD FACS Aria III is 85 MESF for FITC and 29 MESF for PE. The maximum sample acquisition rate is 70.000 events per second. Our system is equipped with 5 laser: wavelength choices include 355, 405, 488, 561 and 633 nm lasers. It measures a maximum of 18 colors simultaneously.