Classification of the Immune Composition in the Tumor Infiltrate
Flow cytometry is one of the most suitable techniques for analyzing and classifying different cell suspensions derived from blood or others compartments. The characterization of all different cellular subtypes is made with different antibodies that detect
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Introduction Fluorescence-activated cell sorting (FACS) is increasingly used worldwide in several areas of clinical and translational research [1, 2], for phenotypic characterization and functional activity by measuring cytokines, intracellular signaling and cell proliferation. It is a rapid, sensitive, quantitative, single-cell analysis technique that still may give impetus to new developments, such as the identification of circulating tumor cells (CTS) [3] or microvesicles [4, 5]. A flow cytometer is constituted of three main systems: fluidics, optics and electronics. The fluidics is the system used for particles transportation, in a single-cell suspension, through the hydrodynamic focusing in front of the laser beam, in the so-called interrogation point. Here cells are enlighten by the lasers and all the characteristics are sent to the optical system, where the fluorescence is divided by band pass filters to select each wavelength detected by photomultipliers (PMT). PMTs are the electronic system converting light signals in electronic signals, for further processing by the computer. Flow cytometry is used for characterization of cells through scatter analysis and fluorescence.
Valentina Proserpio (ed.), Single Cell Methods: Sequencing and Proteomics, Methods in Molecular Biology, vol. 1979, https://doi.org/10.1007/978-1-4939-9240-9_19, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Davide Brusa and Jean-Luc Balligand
Scatter analysis describes the size (forward-scattered light or FSC) and internal complexity (side-scattered or SSC) according to the presence of granules and polylobate nuclei. Fluorescence identifies the different types of white blood cells in lymphocyte, monocyte, macrophage, and granulocyte compartments. Inside the lymphocyte compartment there are many subpopulations, as T, B, NK, T-reg cell subsets that we cannot identify with scatter analysis. Consequently, we need surface markers to identify the specific cell subsets. These markers are called Cluster Designation (CD) and are identified by monoclonal antibodies that in flow cytometry are conjugated by fluorochromes [6]. Fluorochromes are particular molecules that, once irradiated by a laser source, emit fluorescence in a higher wavelength then the excitation one. Emission lights are recorded by the flow cytometer PMTs and data are showed as dots in a Cartesian plane called dot plot. The two axes represent the emission fluorescences and cells are represented as dots according to the amount of fluorescences expressed. Since flow cytometry has always been used to detect different cell subpopulations in different blood diseases, we apply this method to detect immune subpopulations in tumor infiltrates. For this purpose, we process the surgical biopsies of different tumors in order to get a single-cell suspension. Subsequently we stained these samples with a large number of antibodies in order to obtain as much information as possible on cellular subpopulations. Identified cells can be easily isolated with a s
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