Flow Cytometry (FACS)
The ICGEB Flow Cytometry Facility is supplied with three different instruments, for a wide range of possible flow cytometry applications.
Simple analysis of few parameters is performed on the BD Accuri C6 Plus, a cytometer with two excitation lasers (blue, 488 nm, and red, 633 nm) and four fluorescence reading channels at 530 nm (FL1), 585 nm (FL2), 670 nm (FL3) on the blue laser and 675 nm (FL4) on the red laser.
More complex multiparametric analysis can be performed on the BD FACSCelesta cell analyser, equipped with three lasers (violet, 405 nm, blue, 488 nm and red, 633 nm) and the possibility of detecting up to 12 fluorescent channels: 450 nm, 525 nm, 610 nm, 670 nm and 780 nm on the violet laser, 530 nm, 575 nm, 610 nm and 695 nm on the blue laser and 670 nm, 730 nm and 780 nm on the red laser.
Applications of cell sorting can be performed on the BD FACSAria cell sorter, also equipped with three lasers (405 nm, 488 nm and 633 nm) and 13 fluorescence reading channels (450 nm, 510 nm, 610 nm, 660 nm, 710 nm, and 780 nm on the violet laser, 530 nm, 585 nm, 616 nm, 695 nm and 780 nm on the blue laser, and 660 nm and 780 nm on the red laser).
Sorting of selected cells can be achieved with high efficiency and purity (up to 25,000 events/sec for a 4-way sorting with a purity >98%); in addition, the Automatic Cell Deposition Unit allows sorting on slides and tissue culture plates (6, 12, 24, 48, 96 and 384 wells plates are accepted).
We have established optimal conditions for the efficient sterile sorting of CRISPR/Cas9 genome edited cells for subsequent expansion and subcloning. Other possible applications include analysis of gene expression, cell viability and proliferation, cell cycle, ion fluxes in response to specific stimuli, and more.
We offer initial training and support to design panels to identify and isolate rare population out of heterogeneous samples, with a specific focus on the identification of subpopulation of immune cells infiltrating tumours.
Courses are periodically organised, either in presence or from remote, to introduce students to the flow cytometry techniques.
We provide advice and dedicated training sessions in the use of flow cytometry, instrument operation and post-acquisition data analysis on the Flow-Jo software.
More manuals and application procedures can be found in the BD Biosciences site.
Contact persons:
Federica Benvenuti
Group Leader, Cellular Immunology
email: [email protected]
Marco Bestagno
email: [email protected]