BMS2045 2023 Flowcytometry Lecture student view.pdf

Flow cytometry: Principles and Applications Part 1 Dr Kikki Bodman-Smith & Dr Natalie Riddell Session aims Introduce the principles of flow cytometry The application of flow cytometry in Immunology Introduce the problem to be addressed in the flow cytometry practical and how it might be approached u...

Flow cytometry: Principles and Applications Part 1 Dr Kikki Bodman-Smith & Dr Natalie Riddell Session aims Introduce the principles of flow cytometry The application of flow cytometry in Immunology Introduce the problem to be addressed in the flow cytometry practical and how it might be approached using flow cytometry Outline the controls to be used in the above experiment Identifying cells of the immune system Recap The cells of the immune system express molecules on the cell surface many of which are functional components of immune cells These can be used to classify and identify the cells – they are therefore called MARKERS - these are usually on the surface of the cell, but can be expressed internally In 1982, amid growing confusion of alternative names being used, a group of scientists met in Paris to decide on a strict nomenclature for naming these markers: the ‘CD’ system (Cluster of Differentiation) was established and is used today (now there are >370 !) Not all markers/CD molecules are specific for 1 cell type, some may be expressed by a number of different cells …some markers you already know to be associated with leukocytes: Monocyte T cell B cell CD3 CD19 & CD20 no CD19, CD20 or CD14 no CD3 or CD14 CD14 No CD3, CD19 or CD20 We can detect the presence of these markers with Antibodies Recap Antibodies Naïve B cells express Antibodies/membrane Immunoglobulin (IgM and IgD) as part of the surface B cell receptor (BCR) and each B cell against a different antigen When the BCR encounters antigen (and is provided with T cell help) they secrete IgM antibodies against the antigen Later in the immune response, the same antibody variable region may be expressed in IgA, IgG or IgE antibody classes…. Variable regions = Antigen Binding Sites This is known as ‘class switching’ These classes can be further divided into subclasses e.g. IgG 1, 2a, 2b & 3 in mice and IgG1, 2, 3 & 4 in humans which are also called ‘isotypes’ Structure varies slightly between isotypes Constant Region = Effector Function (binding FcRs, activation of complement) Recap Antibodies as scientific tools Commercial Antibody preparations can be produced from one specific B cell (monoclonal Ab: MAb) or from a pool of B cells specific for different epitopes of the same antigen (polyclonal Ab). Can be tagged with fluorescent markers, Enzymes or Radioactive isotopes Antibodies can be used on: cells (single cells, mixtures, tissues) Soluble/secreted/biological products (cytokines, hormones, growth factors) Antibody detection of cell surface (or internal) molecules can be used to: identify/count cells purify cells/tissues/secreted products examine cellular activation/differentiation status examine pathology, expression of abnormalities Techniques that use Antibodies include: Affinity purification Immunoprecipitation ELISPOT Immunofluorescence Histology (immunohistochemistry) ELISA Flow cytometry Streptavidin-HRP ELISA (Enzyme-linked immunosorbant assay) is the detection of secreted soluble products such as cytokines +/- an amplification step with biotin/streptavidin/enzyme linked antibodies Flow cytometry Uses the light scattering properties of single cells in suspension in conjunction with laser excited fluorescence from surface marker specific Antibodies with fluorescent tags (fluorophores) to detect expression of surface or internal markers and or cellular products Biotin Detection Ab Capture Ab Target protein Capture Ab Flow Cytometry Key Features Hydrodynamic or acoustic focussing of a single cell suspension Cell size and granularity are detected by Sheath fluid light scattering patterns Cells can be ‘stained’ or ‘labelled’ for a specific marker with fluorescently tagged antibodies or dyes (fluorophores) Nozzle Fluorophores are excited by a laser, and emit fluorescence at a longer wavelength Fluorescent emissions are filtered, quantified and displayed/stored by the software Multiple, simultaneous measurements Laser light source on the same cell can be made Large numbers of cells can be accurately measured very quickly – also allows for the measurement of rare cell types Sample Hydrodynamic or acoustic focussing Fluorescence emitted from Stained cells Forward and Side scatter light from all cells detected ‘Immunophenotyping’ using flow cytometry Abs specific for cell surface markers on immune cells (CD molecules) can help identify/count/analyse cells By using fluorophores with different excitation/emission profiles, the researcher can MULTIPLEX – use combinations of fluorescently labelled antibodies/dyes to measure multiple cell parameters e.g. Ab to marker A-FITC and Ab to marker B-PE in one tube, allowing the researcher to discriminate between different cell types in the same sample. New fluorophores are continuously being discovered and with increased numbers of lasers, most machines can currently measure up to 12-28 colours, new technology allows