Transcript cell
Separation of the immune competent cells Physical isolation of the cells of interest from a heterogeneous population Differences in the physical , biological or immunological properties of the cells are utilized to separate the cells (differences in cell surface receptor expression is often available) physical – density, size cell biological – adherence, phagocytosis, immunological – antigen differences base strategies: positive separation – labeling and separation of the cells of interest negative separation – get rid of the labeled unwanted cells (depletion) • Important parameters: • purity • recovery, yield Ficoll-Paque: density based cell separation peripherial blood pipettig the „ring” containing the mononuclear cells to a new tube centrifugation To dilute ficoll plasma mononuclear cells (PBMC) ficoll pipetting cells on ficoll, or pipetting ficoll under the cells thrombocytes granulocytes Red blood cells separated cells Isolating or depleting adharent cells Cheap, simple, but only for adharent cells. Low purity and recovery. Antibody ”panning” coated antibodies Complement mediated lysis antibodies complement LYSIS (Red blood cells could be lysated in mild hypotonic ammonium-chloride buffer without any pretreatment) Simple magnetic cell separation Phagocyte cells can uptake small iron particles. These cells could be separated with a strong magnet. Magnetic immunoseparation (MACS) antigene specific antibody paramagnetic bead MACS Magnetic cell separation (MACS) separation of labeled cells (positive separation) CliniMACS – closed system MAGNET MAGNET column depleting or selecting unlabeled cells MicroBeads are very small, usually don’t interfere with cellular functions. CD8+ T cells Flow cytometry Most cells in the immune system can be found in free or loosely adherent form. They can be labeled in cell suspension by fluorescent antigen specific antibodies, and then they can be examined cell by cell. Cells flow in high velocity one by one through a light beam. The light scatter and immunofluorescent properties of the cells are collected and summarized in statistical manner. The method provide qualitative and quantitative data – it can detect the presence of different antigens in the cell, the expression level of this antigen. Changes in the expression of certain molecules can be followed after different treatment of the specimen. Example Chanel Layout for Laserbased Flow Cytometry The emited fluorescent light can be separeted to components by special mirrors and filters photodetectors PMT 4 cell suspension in tube flow cell forward light scatter detector PMT 3 pl. PE PMT 1 PMT 2 pl. FITC side light scatter detector Laser (PMT=photo-multiplayer tube) Fluidics System sheet fluid reservoir Flow cell Injector +++ +++ +++ sheet fluid sample 6-10m/s flow rate Fluorescent light Focused laser beam Fluorescence Laser FALS Sensor FSC Fluorescence detectors (PMT3, PMT4 etc.) autofluorescence - piridins and flavins Becton Dickinson cytometers from the ’80-ies and ’90-ies benchtop flow cytometer high speed sorter-flow cytometer (FACS, FACS station) 30,000 cells/sec Characterisation of immune cells using cell surface markers Cell types, differentiation stages can be identified using a combination of cell surface markers. Used in diagnostics: - ratio of different cell types - altered expression of cell surface markers Examples: - Inflammatory processes – increased neutrophil numbers - HIV progression – decrease of CD4+ T cell count CD4+ : CD8+ = 1.6 Normal CD4+ T cell count = 600 – 1400/l AIDS = CD4+ T cell count <200/l - increase of CD5+ B cells – typical for some B cell Leukemias WAS: Wiscott-Aldrich Syndrome Lack of CD43 expression XLA: X-linked Agammaglobulinemia Inhibited B cell development lack of CD19 CD antigen cell type function ligand CD3 T cells TCR signalling - CD4 helper T sejtek, (monocytes, T cell coreceptor, (HIV receptor) MHC- II, HIV pDC) CD5 T cells, (B cell subset: B1) adhesion, activation signals CD72 CD8 cytotoxic T cells, (NK, T cells) T cell coreceptor MHC I CD14 monocytes, macrophages, some granulocytes LPS binding LPS, LBP CD19 B cells part of CR2, B cell coreceptor C3d, C3b CD28 T cells costimulatory signals to T cells (B7-1, B7-2) CD80, CD86 CD34 hematopoietic progenitor cell adhesion CD62L (L-selektin) CD56 NK cell, (T and B cell subset) homoadhesion (N-CAM isoform) APC: DC, B, monocyte, macrophage costimulatory signals CD80, CD86 (B7-1, -2) CD28, CD152 Different cell types - characteristic light dispersions granulocytes side light dispersion (SSC) (e.g. granulated) monocytes lymphocytes forward light dispersion (FSC) („size”) The method for erythrocyte exemption affects the distribution of remaining cell types Lysis of erythrocytes Ficoll-Paque density-based separation missing granulocytes ”Gating” of different cell populations granulocyte „gate” monocyte „gate” lymphocyte „gate” Immunophenotyping Example: Measurement of CD4+ (helper) and CD8+ (cytotoxic) T cell ratio (eg. monitoring AIDS progression) (based on primari antibody-antigen reactions) Labeling: FITC labeled anti-CD4 antibody(α-CD4-FITC) PE labeled anti-CD8 antibody (α-CD8-PE) Th NK Tc Lymphocytes in the periferial blood B detecting CD4-FITC labeled (TH) cell high velocity flow stream (in cuvette or stream in air) detector signal processing unit CD8 PE Screen increasing light intensity a dot representing a CD4+ CD8- cell CD4 FITC detecting the PE labeled cell (CD8-PE) CD8 PE detector signal processing unit increasing light intensity CD4 FITC detecting the unlabeled cell (eg.B cell) by autofluorescence CD8 PE detector jelfeldolgozó signal processing egység unit CD4 FITC CD8 PE 18% 44% 0% quadrant statistics CD4 38% FITC Graphical representations 1. dot-plot contourplot densityplot Graphical representations 2. Histogramm FACS (Fluorescence Activated Cell Sorting) any distinctive cell population can be gated, and the gated cells could be separated Different gating strategies can be combined: • by light scattering • by specific immunofluorescence PMT 4 Sample PMT 3 Flow cell PMT 2 PMT 1 Laser Example: •CD5+ B1 cell separation (CD19/CD5) •NKT cell separation (CD3/CD56) NKT cells NK cells lymphocytes The fluid stream break up into dropplets by the vibration of the flow cell. breakoff point vibration (nozzle orifice of the flow cell) + + + + + + + + + Laser + charged deflection + plate + + If the wanted cell reach the breakoff point, the stream become charged for the short time of drop formation, and the formed drop become charged + + + + + - charged deflection plate - --- collection tube collection tube waste