Project

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Multicolor flow cytometric analysis of the immune system during homeostasis and activation

Applicant Luther Sanjiv
Number 128808
Funding scheme R'EQUIP
Research institution Département de Biochimie Faculté de Biologie et Médecine Université de Lausanne
Institution of higher education University of Lausanne - LA
Main discipline Immunology, Immunopathology
Start/End 01.01.2010 - 31.12.2010
Approved amount 235'300.00
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All Disciplines (2)

Discipline
Immunology, Immunopathology
Experimental Cancer Research

Keywords (11)

multicolor flow cytometry; lymphocytes; stromal cells; dendritic cells; hematopoiesis; stem cells; cellular activation; FRET; cell cycle; Immune system;

Lay Summary (English)

Lead
Lay summary
Research in Immunology strongly relies on multi-parameter flow cytometric analysis that measures the phenotype and function of the various immune cells found in blood, lymphoid organs or other sites. This technology is highly quantitative and enables the precise analysis of heterogeneous and very large cell populations with single cell resolution. Over the last few years this technology has rapidly advanced and now permits the simultaneous analysis of cellular states or processes together with surface proteins in multiple fluorescent colors. Thus it provides scientists with a unique analysis platform giving profound insights into the composition and status of the immune system.Multi-parameter flow cytometry relies on the principle that different immune cell types can be distinguished by using fluorochrome-coupled antibodies to specific proteins expressed at the surface of some but not other immune cell types. A flow cytometer is capable of visualizing these fluorochrome-labelled cells (similar to a fluorescence microscope), except that the cells pass through the machine in a tiny fluid stream at a speed of up to 20'000 cells per second and many more fluorescent parameters as well as physical characteristics such as size, shape and internal complexity can be determined. This particular flow cytometer (5 laser LSR II with 20 detectors) can simultaneously measure 18 different fluorochromes, using the remaining detectors for cell size and shape. Moreover, these parameters can be measured on millions of cells at the single cell level. The possible applications of this highly quantitative machine include not only multicolor surface staining (differential phenotype), but also the simultaneous analysis of intracellular protein expression, Ca2+ flux and DNA content. Thereby insights can simultaneously be gained into the metabolic activity, cell cycle status, cell death, cellular turnover status, intracellular signaling, cytokine production and several other aspects (phenotypic and functional analysis of cells). The Epalinges research campus currently has 15 Immunology laboratories with 10 more immunology laboratories at the close by hospital, most of which use flow cytometry to analyze the outcome of their experiments. No equivalent machine is currently owned by the University of Lausanne. Therefore, this machine will greatly help in the research efforts of many local Immunology laboratories and will allow new types of analysis. This high-end machine should significantly enhance the competitiveness of the Epalinges research campus and strengthen the prominence of Immunology research in Switzerland.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Associated projects

Number Title Start Funding scheme
130488 Fibroblasts of secondary lymphoid organs: central players in tissue homeostasis and immunity 01.05.2010 Project funding (Div. I-III)
116896 T zone stromal cells in secondary lymphoid tissues and their roles in dendritic cell retention and T cell homeostasis 01.08.2007 SNSF Professorships
166161 Fibroblasts of lymphoid tissues: from phenotype to function 01.05.2016 Project funding (Div. I-III)
146944 Fibroblasts of secondary lymphoid organs: characterization of their development and function 01.05.2013 Project funding (Div. I-III)

Abstract

Research in immunology strongly relies on multi-parameter flow cytometry to measure the phenotype and function of immune cells. This technology is highly quantitative and enables the analysis of mixed cell populations with single cell resolution. Moreover, it permits easy detection of rare cell populations. Over the last few years this technology has rapidly advanced and now allows the simultaneous analysis of cellular states or processes together with surface proteins in up to 18 fluorescent colors. Staining of multiple surface markers to identify hematopoietic cell subsets can now be combined with intracellular staining for cytokine production, signal transduction and activation molecules, phosphorylation status and Ca2+ flux to obtain functional readouts. In addition, certain physical properties of different cell types, such as side population ability, metabolic activity, cell cycle, DNA content (proliferation versus death) and turnover status can also be assessed in combination with surface markers. Moreover, gene technology frequently requires the detection of fluorescent proteins that may have been integrated into the genome as genetic markers. All this requires state-of-the art flow cytometers with the capacity to measure 9 or more colours, and at the same time Ca2+ flux or cell cycle status. Finally, flow cytometry can be used to detect intra-molecular florescence energy transfer (FRET) in order to measure the proximity between two proteins carrying different fluorochrome tags. The success of this technique depends on the appropriate excitation wavelength and the ability to detect a well-defined emission signal as only specific combination of fluorochromes are applicable for this technique. The projects described in this grant proposal all utilize (and are critically dependent on) one or more of these advanced technologies. Four of the 6 projects (Luther, Acha-Orbea, Tschopp and Wilson) projects use multi-parameter surface staining to characterize the highly heterogeneous cell subsets found in primary or secondary lymphoid organs, both during normal development, homeostasis or immunity. The same approach is used to identify and characterize cell subsets found in non-lymphoid organs. To understand these processes in more detail cells from mice deficient in key gene products can be analyzed for changes in their relative proportion or metabolic state of different cell populations. Most of these projects also depend on the ability to simultaneously detect surface molecules together with intracellular molecules (cytokines, TCRs) or intrinsic cell properties such as apoptosis and cell cycle that can be modified in the absence or overexpression of many different types of genes. By directly combining surface staining with cell cycle analysis or Annexin V/Hoechst staining, these cellular properties can by easily assessed on a per cell basis. This will not only significantly enhance experimental results, but it should also reduce both the number of animals and expensive reagents required for individual experiments. Given that this machine can analyze up to 20 parameters (18 fluorochromes plus forward and side-scatter characteristics) at 20’000 cells per second, significant savings in time analyzing cells can be achieved. In addition, the statistical significance of rare cell population analysis can be dramatically improved over data obtained with conventional flow cytometers.Three of the projects (Luther, Acha-Orbea and Wilson), will exploit this machine’s ability to analyze rare cell populations such as stem and progenitor cells from the bone marrow, stromal cells and dendritic cells from secondary lymphoid organs. In all cases, multiple surface markers are required just to define each subset, then functional properties of these cells can be measured with the extra fluorescent parameters that can be detected by this machine. These include intracellular cytokines and signaling proteins, DNA and RNA content, expression of other intranuclear cell cycle proteins (for example Ki67), and molecules involved in apoptotic processes such as granzymes, Annexin V and Tunnel staining. Reporter gene expression is frequently by fluorescent proteins like GFP, YFP, etc., so this often increases the minimum number of parameters required to more than 9. Cross correlation of the flow cytometry with results obtained with immunohistochemistry will provide further information concerning the localization of rare subsets in different tissues. The same flow cytometric techniques will be applied to the analysis of normal and genetically modified cell lines.Two of the projects (Thome and Luescher) will use the same experimental flow cytometric technique (FRET) to study completely different immunological questions. In the first, the regulatory mechanism of MALT1 proteolytic cleavage activity in living cells will be studied. FRET between 2 fluorescent fusion proteins (CFP and YFP) linked by a MALT1 specific cleavage sequence is lost upon MALT1-dependent cleavage. In the second, FRET will be used to measure the physical proximity between components of the TCR/CD3/CD8 complex or between the 2 chains of the CD8 heterodimer itself (on the surface of T cells).A common technique used to study cellular activation on a per cell basis is Ca2+ flux, which exploits the wavelength changes of Indo1 detected by a UV laser. This will be used to detect the molecular mechanisms underlying inflammasome activation (Tschopp), or to quantitate different pathways of activation in T cells (Luescher). All of these proposed projects are dependent on the use of a LSR II flow cytometer. The machine will be equipped with 5 lasers: 4 lasers used for maximum multi-colour analysis, the UV laser for cell cycle and Ca2+ flux detection, the violet laser for FRET. It will satisfy the current and future needs of the large immunology community present on the Epalinges research campus.
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