Presentation
The cytometry platform provides the scientific community with state-of-the-art analyzer and sorting cytometers. It also offers the expertise of its staff for the development of experimental protocols or new applications in cytometry, multi-color panels, and assistance for the analysis of cytometry data.
The cytometry platform is open to all research institutes and private companies.
The flow cytometry technique allows the multi-parametric analysis, very quickly, of individualized cells in suspension within a sample which may consist of different cell populations, even extremely rare. Sorting cytometers are capable of separately collecting identified cell subpopulations, or depositing a specific single cell into each well of 96- or 384-well plates for cloning or single-cell level analyses.
Location
Plateforme de cytométrie de la SFR Necker
Faculté de Médecine Université de Paris
156-160 rue de Vaugirard
75015 Paris
Tél : 01 40 61 54 73
The SFR Necker cytometry platform is at 1st flour of the Faculty building
Staff
IE, Inserm
Sara Rohou
AI-CDD
Equipements
The platform is equipped with 4 sorting cytometers and 2 analyzing cytometers including a spectral analyzer, and provides a laboratory dedicated to the cell sorting of infectious samples (pathogenic bacteria and viral infections).
The Sony ID7000 spectral analyzer cytometer can simultaneously analyze 41 fluorochromes for each cellular sample. It is equipped with an automatic sampler for 96 and 384 well plates and 5ml tubes and sample temperature control.
The sorters (except the Sony SH800) are equipped with a depositing machine which sorts and deposits a predefined number of cells in multi-well cell culture plates (96 or 384 wells), strips of micro - PCR tubes or microscope slides. Sorts in Single-Cell mode allow you to deposit a single cell in each well. The “Index Sorting” option makes it possible to identify the cells deposited in each well.
Sony SH800
3 lasers : 488, 561 and 640nm
6 fluorescences can be detected simultaneously
70, 100 and 130um nozzles
Sorting speed is 12,000 cells per second
Services
Assisted services: The user is assisted by the platform staff for his experimental adjustments, or if the use of cytometers is infrequent.
Unassisted services: Platform staff train users on all cytometers to become self-sufficient.
The platform also provides data analysis software such as Diva, Sony, FlowJo, Kaluza, but also high-dimensional analysis by dimensionality reduction of complex multi-color data: OMIQ
Applications in cytometry:
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phenotyping, membrane and intracellular multi-markings
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analysis of cells transfected with fluorescent reporter systems (GFP, mCherry, etc.)
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analysis of “Side Population” hematopoietic stem cells (Hoechst 33342)
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mono-parametric cell cycle study (Permeabilized cells: IP, Intact cells: Hoechst33342), multi-parametric cell cycle study (Incorporation of nucleosides: BrdU, EdU, RNA labeling with Pyronin), cell cycle study with the colored indicators (red and green) genetically encoded FUCCI
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analysis of cell death by apoptosis
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study of the cell's vital parameters: cell proliferation (CFSE), mitochondrial membrane potential (JC-1, DiOC2, MitoTracker), enzymatic activity, membrane integrity (IP, Sytox dyes, 7-AAD), study of signaling pathways ( phospho-proteins)
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multiplex assays (cytokines) with the BD Cytometric Bead Array
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study of calcium flux (Indo-1)
The platform can develop new applications according to the technological potential of the equipment and their possible evolution.
Publications
Direct contribution of skeletal muscle mesenchymal progenitors to bone repair. Julien A, Kanagalingam A, Martínez-Sarrà E, Megret J, Luka M, Ménager M, Relaix F, Colnot C. Nat Commun. 2021 May 17;12(1):2860.
Maturation and persistence of the anti-SARS-CoV-2 memory B cell response. Sokal A, Chappert P, Barba-Spaeth G, Roeser A, Fourati S, Azzaoui I, Vandenberghe A, Fernandez I, Meola A, Bouvier-Alias M, Crickx E, Beldi-Ferchiou A, Hue S, Languille L, Michel M, Baloul S, Noizat-Pirenne F, Luka M, Mégret J, Ménager M, Pawlotsky JM, Fillatreau S, Rey FA, Weill JC, Reynaud CA, Mahévas M. Cell. 2021 Mar 4;184(5):1201-1213.e14.
Toll-like receptor-9 stimulated plasmacytoid dendritic cell precursors suppress autoimmune neuroinflammation in a murine model of multiple sclerosis. Letscher H, Agbogan VA, Korniotis S, Gastineau P, Tejerina E, Gras C, Mégret J, Moe A, Drobyski WR, Zavala F. Sci Rep. 2021 Feb 26;11(1):4735.
Hepatitis B virus X protein promotes DNA damage propagation through disruption of liver polyploidization and enhances hepatocellular carcinoma initiation. Ahodantin J, Bou-Nader M, Cordier C, Mégret J, Soussan P, Desdouets C, Kremsdorf D. Oncogene. 2019 Apr;38(14):2645-2657.
A splenic IgM memory subset with antibacterial specificities is sustained from persistent mucosal responses. Le Gallou S, Zhou Z, Thai LH, Fritzen R, de Los Aires AV, Mégret J, Yu P, Kitamura D, Bille E, Tros F, Nassif X, Charbit A, Weller S, Weill JC, Reynaud CA. J Exp Med. 2018 Aug 6;215(8):2035-2053.
BAFF and CD4+ T cells are major survival factors for long-lived splenic plasma cells in a B-cell-depletion context. Thai LH, Le Gallou S, Robbins A, Crickx E, Fadeev T, Zhou Z, Cagnard N, Mégret J, Bole C, Weill JC, Reynaud CA, Mahévas M. Blood. 2018 Apr 5;131(14):1545-1555.
Periosteum contains skeletal stem cells with high bone regenerative potential controlled by Periostin. Duchamp de Lageneste O, Julien A, Abou-Khalil R, Frangi G, Carvalho C, Cagnard N, Cordier C, Conway SJ, Colnot C. Nat Commun. 2018 Feb 22;9(1):773.
Treatment of ongoing autoimmune encephalomyelitis with activated B-cell progenitors maturing into regulatory B cells. Korniotis S, Gras C, Letscher H, Montandon R, Mégret J, Siegert S, Ezine S, Fallon PG, Luther SA, Fillatreau S, Zavala F. Nat Commun. 2016 Jul 11;7:12134.
A unique CD8(+) T lymphocyte signature in pediatric type 1 diabetes.
Hamel Y, Mauvais FX, Pham HP, Kratzer R, Marchi C, Barilleau É, Waeckel-Enée E, Arnoux JB, Hartemann A, Cordier C, Mégret J, Rocha B, de Lonlay P, Beltrand J, Six A, Robert JJ, van Endert P. J Autoimmun. 2016 Sep;73:54-63.
Lymphoid Gene Upregulation on Circulating Progenitors Participates in Their T-Lineage Commitment. Zepponi V, Michaels Lopez V, Martinez-Cingolani C, Boudil A, Pasqualetto V, Skhiri L, Gautreau L, Legrand A, Megret J, Zavala F, Ezine S. J Immunol. 2015 Jul 1;195(1):156-65.
Innate pro-B-cell progenitors protect against type 1 diabetes by regulating autoimmune effector T cells. Montandon R, Korniotis S, Layseca-Espinosa E, Gras C, Mégret J, Ezine S, Dy M, Zavala F. Proc Natl Acad Sci U S A. 2013 Jun 11;110(24):E2199-208.