Central Support Units for Biomedical Research

Flow Cytometry Unit

The mission of the Unit is to offer access to high performance equipment that covers a broad spectrum of applications in the field of Flow Cytometry to IMIBIC and hospital setting researchers, and to other public institutions and private sector, as well as to provide technical expertise and training. The Unit also provides scientific-technical advice on experimental design, sample preparation, Cytometry data analysis and interpretation of results.

The Flow Cytometry unit has a Quality Management System in accordance with the ISO 9001:2015 Quality Standard.

Personnel

Fundamentals and applications of Flow Cytometry

Flow Cytometry is a powerful technique that simultaneously and rapidly measures and then analyzes multiple physical and/or chemical characteristics (structural or functional) of single particles in suspension, usually cells, as they flow in a fluid stream through a beam of laser light. The determination of these characteristics is based on measurements of incident laser light scattering and emitted fluorescence from each particle or cell using an optical-to-electronic coupling system.

Fluorescence-activated Cell Sorting (FACS) is a specialized type of Flow Cytometry. It provides a method for separating and isolating cell subpopulations from heterogeneous mixtures, one cell at a time, based on the light scattering and fluorescent characteristics of each cell.

APPLICATIONS

There is a wide range of flow cytometry applications, including, but not limited to:

• Multicolour analysis of cell surface and intracellular antigens using fluorochrome-labeled antibodies
• Cell viability
• Apoptosis assays (plasma membrane integrity, mitochondrial function, caspase activity, DNA fragmentation, etc.)
• DNA content analysis and cell cycle
• Functional assays, such as cell proliferation (e.g. CFSE), ROS production, membrane potential measurements, Calcium flux, intracellular pH measurements, etc.
• Gene expression analysis by using reporter genes
• Flow-FISH and PrimeFlow RNA assay
• Microvesicles analysis
• Microorganism analysis
• Separation and isolation of specific cell subpopulations or single cells, and cell cloning by FACS Cell Sorting

Equipment and facilities

Analyzers

• BD LSRFortessa SORP (Becton Dickinson) (Last update: 2021): equipped with 5 lasers (355 nm, 405 nm, 488 nm, 561 nm and 640 nm) and capable of analysing up to 20 parameters simultaneously, 18 fluorescence parameters plus forward and side scatter. (optical configuration)
• BD FACSCanto II (Becton Dickinson) (Last update: 2021): equipped with 3 lasers (405 nm, 488 nm and 633 nm). It allows detection of 10 parameters simultaneously, 8 fluorescence parameters plus forward and side scatter. Equipped with Multi Carousel Loader (up to 40 tubes). (optical configuration)
• BD FACSCalibur (Becton Dickinson): equipped with 1 Argon laser (488 nm). It allows detection of 5 parameters simultaneously, 3 fluorescence parameters plus forward and side scatter. (optical configuration)

Cell sorter

• BD FACSAria III (Becton Dickinson)(Last update: 2014). (optical configuration)

Features:

• Equipped with 3 lasers (407 nm, 488 nm and 633 nm), it allows to analyze up to 13 parameters simultaneously, 11 fluorescence parameters plus forward and side scatter.
• Available nozzles: 70µm, 85µm and 100µm.
• Four-way and single-cell sorting capabilities. Sorted cells can be collected into tubes (microtubes, 12 x 75 mm and 15 mL), plates (6, 24, 48, 96 and 384 wells), and onto slides (equipped with ACDU).
• Temperature Control: sample input (software-adjustable) and sample output for sort collection (water recirculation unit).
• Equipped with Aerosol Management Option (AMO).

Others

• PC workstation with Flow Cytometry data analysis software BD FlowJo v10.

Services

• Self-use (Operator-unassisted) of flow cytometry analyzers (only for IMIBIC users properly trained by us to use the equipment).
• Operator-assisted use of the analyzers on demand.
• Cell sorting: only provided as an operator-assisted service.
• Flow cytometry data analysis on demand. The unit also offers registered users access to PC workstation with data analysis software BD FlowJo.
• Advice on experimental and multicolor panel design, sample preparation methods, interpretation and presentation of data and writing of the “material and methods” section of Flow Cytometry.
• Developing/implementing Flow Cytometry assays/applications responding to the user needs.

FEES