Prof. Oberdan LEO, Ph. D., ULB IBMM, IMI
oleo@ulb.ac.be
Professor Oberdan Leo is Professor at the Université Libre de Bruxelles (ULB), and Assistant Professor at the Université de Mons (UMons) Belgium, where he teaches Immunology. Since 1999, he has served as President of the Belgian Immunological Society. Professor Leo's
major research interests focus on the relationship between metabolism and the inflammatory response and the analysis of T helper subset differentiation pathways. He is presently Director of Institute for Medical Immunology, a research institute dedicated to Clinical and Experimental Immunology created in 2004 thanks to a joint public -private research program initiated by GSK Biologicals, ULB and the Walloon Region.
Adrien GALUPPO : Flow Cytometry (ImageStream, Cellular Sorting)
agaluppo@ulb.ac.be
Adrien Galuppo was born in September 1984. He obtained his Master degree in Industrial Engineer in Biochemistry from the HEPCUT – Condorcet (Charleroi) in 2009. In the Institute for Molecular Biology and Medicine of Université Libre de Bruxelles, he completed his training in the team of Muriel Moser (Immunobiology laboratory) where he acquired technical expertise in Immunology. From August 2009 to August 2012, he worked in the Immunobiology lab where he is responsible for cell sorting by flow cytometry and he provided technical assistance in cellular culture, histology, flow cytometry, microscopy, etc. From August 2012, he joined the CMMI to provide technical assistance on ImageStream flow cytometry.
Imaging Flow Cytometry
Equipment
The device consists of an Imaging Flow Cytometer ImageStream 100 (Amnis, Seattle) (Figure 1). It allows to acquire several different images per cell and light illumination is provided by 3 laser sources (violet at 405 nm, blue at 488 nm and red at 658 nm) as well as a brightfi eld source.
Figure 1 - ImageStream 100 (Amnis, Seattle)
Figure 2 - Localisation of transcription factor NF-κB in T lymphocyte cells before (A) and after (B) anti-CD3 stimulation. Panels (from left to right), show images recorded with brightfi eld source (BF), NF-κB, nuclear marker Draq5 and the composite view of NF-κB and Draq5.
Bibliographical references
Salmon D, Vanwalleghem G, Morias Y, Denoeud J, Krumbholz C, Lhommé F, Bachmaier S, Kador M, Gossmann J, Dias FB, De Muylder G, Uzureau P, Magez S, Moser M, De Baetselier P, Van Den Abbeele J, Beschin A, Boshart M, Pays E. Adenylate cyclases of Trypanosoma brucei inhibit the innate immune response of the host. Science. 2012, 337:463-6.
Schillewaert S, Wacheul L, Lhomme F, Lafontaine DL. The evolutionarily conserved protein Las1 is required for pre-rRNA processing at both ends of ITS2. Mol Cell Biol. 2012, 32:430-44.
Adams B, Dubois A, Delbauve S, Debock I, Lhommé F, Goldman M, Flamand V. Expansion of regulatory CD8+ CD25+ T cells after neonatal alloimmunization. Clin Exp Immunol. 2011, 163:354-61.
Vokaer B, Van Rompaey N, Lemaître PH, Lhommé F, Kubjak C, Benghiat FS, Iwakura Y, Petein M, Field KA, Goldman M, Le Moine A, Charbonnier LM. Critical role of regulatory T cells in Th17-mediated minor antigen-disparate rejection. J Immunol. 2010, 185:3417-25.
De Wilde V, Van Rompaey N, Hill M, Lebrun JF, Lemaître P, Lhommé F, Kubjak C, Vokaer B, Oldenhove G, Charbonnier LM, Cuturi MC, Goldman M, Le Moine A. Endotoxin-induced myeloid-derived suppressor cells inhibit alloimmune responses via heme oxygenase-1. Am J Transplant. 2009, 9:2034-47.
Benghiat FS, Craciun L, De Wilde V, Dernies T, Kubjak C, Lhomme F, Goldman M, Le Moine A. IL-17 production elicited by allo-major histocompatibility complex class II recognition depends on CD25posCD4pos T cells. Transplantation. 2008 85:943-9.
Application
The Flow Cytometry Core Facility offers access to two analysers, both equipped with 3 lasers (405 nm, 488nm, and 640 nm) and respectively 9 (Beckman Coulter Cyan ADP cytometer) and 14 (BD LSR Fortessa cytometer) fluorescence detectors for multi-parametric analyses. The platform is also equipped with a high speed cell sorter (BD Facs Aria) allowing the simultaneous separation of up to four cell populations. The platform also provides magnetic cell sorting and has access to the Luminex technology (Biorad bioplex 200) enabling the simultaneous analysis of up to 100 different biomolecules per sample.
Description
Imaging Flow Cytometry is a technology which combines in one single device the advantages of the statistical rigour of fl ow cytometry with those of the visual power of microscopy. ImageStream enables fast acquisition of images of cells in a fl ux and allows to analyse them both qualitatively and quantitatively. The analysis is based on criteria such cell morphology, location or intensity of markers on, in or between cells.
The IFC method has been applied to carry out functional studies, phenotyping, cell cycle, apoptosis/nuclear damage, transcription factor nuclear translocation (Figure 2). It has also been usedin a whole range of different disciplines, including immunology, microbiology, parasitology, vaccinology, or cell therapy.
See the poster: POSTER_CMMI_-_Imaging_Flow_Cytometry.pdf
