Central Support Units for Biomedical Research

Flow Cytometry Unit

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

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:

• Phenotypic analysis by surface and intracellular antigen staining.
• Cell viability and 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.
• Microorganism analysis.
• Small particle analysis.
• Separation of cell subpopulations from heterogeneous samples and single-cell isolation by 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)

Cell sorters

• 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).
• Equipped with Aerosol Management Option (AMO).

Data analysis Software

• 1 offline BD FlowJo v10 license (PC workstation).

Services

• Self-use (Operator-unassisted) of analysers (only for IMIBIC users properly trained by us to use the equipment).
• Operator-assisted use of analysers on demand.
• Cell sorting: only provided as an operator-assisted service.
• Flow Cytometry data analysis on demand.
• Advice on experimental and multicolour panel design, sample preparation methods, data interpretation and presentation, and drafting the "Materials and Methods" section for Flow Cytometry.
• Developing and implementing Flow Cytometry assays/applications tailored to user needs.