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Detection of intracellular antigens at the single cell level using flow cytometry Niga Nawroly Imperial College London [email protected] HOW? Surface Phenotyping + Intracellular + How does a flow cytometer work? Electronics Fluidics Light detectors FL3 PerCP, Cy5 FL2 PE FL1 FITC SSC Laser Light 488nm FSC Computer Detection of Intracellular proteins: Studying Intracellular Enzyme Activity • Enzymes are found in all cell types. • Involved in almost every cellular process. • Cyclooxygenase (COX) • Terminal Deoxynucleotidyl Transferase (TdT) • Myeloperoxidase (MPO) Fixation does not allow the detection of active enzyme in a cell. Detection of Intracellular proteins: Signal transduction Important in distinguishing changes in signaling status that arise in cells during functional activation. • Phosphorylated STAT-1 • KINASES (p38 MAPK,P44/42 MAPK, JNK/SAP). • Members of cell survival pathways (AKT/PKB) • T cell activation pathway (TYK2) • Anti Phosphorylated ERK Detection of Intracellular proteins: Immunological responses Inflammatory mediators such as perforin or granzyme. Cytokines and Chemokines Cytokines Cytokines are small secreted proteins produced by lymphocytes. • Mediate and regulate immunity, inflammation, and hematopoiesis. • Produced in response to an immune stimulus. • They bind to specific membrane receptors. Cytokine Production Signal Cytokines Picture From www.fredonia.edu LPS Ionomycin PHA T cell Con A CD3 CD28 To enhance the accumulation of intracellular cytokines. Stimulation Monensin: Cytokines accumulate in the ER Brefeldin A: in Golgi complex. Secretion stop (Brefeldin A or Monensin) • Polyclonal stimulation Only in vitro • Specific, monoclonal stimulation… Peptide, Virus, Bacteria To maintain structural integrity. Formaldehyde or glutaraldehyde Keep the protein structure and doesn't change the Saponin (permeablisation buffer). (accessibility of the) epitopes too much Intracellular Staining Permeabilisation Fixation Multiparameter Intracellular technique Two or more antibodies!... To analyse multiparameter data at the level of one cell • Cell size and granularity • CD antigens • Cytokine receptors • Adhesion molecules • Migration/Homing molecules • Cell cycle, G0, G1, S, G2 Combination of surface and intracellular flow cytometry. 1 Colour 2 Colour Advantages of Multi-colour 4 2 1 3 Colours 2 Population 12 2 3 3 1 4 Colours 5 Colours 6 Colours 1 2 24 40 60 ? Which fluorochromes and when … ANTIGEN DENSITY FLUOROCHROME low Phycoerythrin, APC low-intermediate CY5 high FITC, PerCP What dye? Problems Non-specific binding (false +Ve result) Fc receptor blocking, Mouse systems, FcγII/III CD16/CD32 Human, human Ig or 10% autologous serum in PBS Fc Receptor Anti-Fc Receptor Problems Non-specific binding (false +Ve result) Immunoglobulin Isotype Controls Same fluorochrome-conjugated antibody of irrelevant specificity which has the same Ig isotype. Ligand blocking control Pre-block with anti-cytokine antibody. Problems Autofluorescence Run an unstained sample Dead cells Centrifugation, Ficoll sepaeration, or by using propidium iodide. Can be detected on Forward Scatter channel…. Spectral Overlap Single-stained cells. Apply electronic compensation Problems Spectral overlap FL-2 PMT FL-1 PMT FL-1 -% FL-2 FL-2 -% FL-1 Electronic compensation 10 4 10 11 PE 10 2 PE 10 2 10 3 10 4 11 10 3 10 R10 CD8 10 1 10 1 R10 12 13 10 1 10 2 APC 10 3 10 4 RANTES Lung BALB/c Jonathan Dodd 12 13 10 1 10 2 APC 10 3 10 4 4 10 4 15 10 11 IL-5-APC 10 2 CD3-QR 10 2 10 10 3 3 10 14 CD3 16 R10 17 10 1 2 10 DX5-PE 10 3 10 10 IL-5 R14 1 1 NK T 10 12 4 13 10 1 INF-g DX5 BAL BALB/c Wieslawa Olszewska 10 2 IFNg-FITC 10 3 10 4 Data Analysis Available analysis software • Cellquest • WinList • Flowjo • WinMDI Free cytometry software: http://flowcyt.cyto.purdue.edu/flowcyt/software/Catalog.htm Flow Cytometry on the web • ISAC www.isac-net.org • Salk Institute http://flowcyt.salk.edu • Purdue University www.cyto.purdue.edu • Scripps Research Institute http://facs.scripps.edu/index.html • CRUK http://science.cancerresearchuk.org/sci/facs Our website http://www.wfi.med.ic.ac.uk/Flow.htm New Website address: www1.imperial.ac.uk/medicine/about/institutes/wfi/flow Our facility provides: • BD FACS LSR machine (analyser). Three-laser, Six-colour benchtop flow cytometer. • BD FACS Calibur machine (analyser). One laser (488-nm) four-colour multipurpose flow cytometer. • BD FACSDiva machine (cell sorter). Three-laser, Six-colour cell sorter. Our facility provides: • Luminex Xmap, multi parameter array. • ELISPOT reader, to measure AntigenSpecific Cytokine Production of Single Cells. plus stand-alone analysis computers that can be used for off-line analysis. www1.imperial.ac.uk/medicine/about/institutes/wfi/flow [email protected]