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8th SEMINAR

8th SEMINAR. SEPARATION AND MEASUREMENT OF THE ACTIVITY OF IMMUNECOMPETENT CELLS. CELL SEPARATION. Physical isolation of the cells of interest from a heterogeneous population

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8th SEMINAR

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  1. 8th SEMINAR SEPARATION AND MEASUREMENT OF THE ACTIVITY OF IMMUNECOMPETENT CELLS

  2. CELL SEPARATION 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 – there is a possibility to further investigate the separated living cells). • physical – density, size • cell biological – adherence, phagocytosis, sensitivity to the medium • immunological – antigen differences (surface marker) Consideration taken to: purity, recovery, yield and viability of the cells

  3. TWO SEPARATION STRATEGIES Positive separation Negative separation Labeled the unwanted cells (depletion) The cells become affected only by the separation environment, hence this is the preferred strategy in functional examinations. Labeling and separation of the cells of interest e.g. labeling a cell surface molecule by a fluorescent antibody. The cells become affected both by the separation environment and the antibodies bound to the receptors. The purity of the separation is generally high.

  4. peripheral blood (orbuffycoat) centrifugation plasma mononuclear cells (PBMC) ficoll Neutrophil granulocytes separated cells pipetting cells on ficoll Red blood cells FICOLL-PAQUEDENSITY BASED CELL SEPARATION pipettig the „ring”containing the mononuclear cells to a new tubeto get rid of Ficoll

  5. (fromGooglepictures) (NatureProtocols http://www.nature.com/nprot/journal/v3/n6/images/nprot.2008.69-F1.jpg)

  6. paramagnetic bead SEPARATION METHODS BASED ON THE IMMUNOLOGICAL PROPERTIES OF THE CELLS Magnetic-Activated Cell Sorting (MACS) antigen specific antibody

  7. MAGNET MAGNET column depleting or selecting unlabeled cells (negative separation)

  8. NKT cells NK cells lymphocytes SEPARATION METHODS BASED ON THE IMMUNOLOGICAL PROPERTIES OF THE CELLS Fluorescence-Activated Cell Sorting (FACS) Example: NKT cell separation (CD3/CD56) blood sample T cells

  9. The fluid stream break up into droplets by the vibration of the flow cell. breakoff point

  10. + + + + + + + + + vibration (nozzle orifice of the flow cell) Laser If the wanted cell reaches the breakoff point, the stream become charged for the short time of drop formation, so the formed drop become charged - + - + charged deflection plate charged deflection plate + + + - + + + + - - - - - - collection tube collection tube waste

  11. MEASURING THE ACTIVITY OF IMMUNECOMPETENT CELLSPHAGOCYTIC CELLS – PHAGOCYTOSIS ASSAY • Using killed pathogens (bacteria: E. coli, S. aureus; yeast: S. cerevisiae) labeled with different fluorophores • Phagocytosis can be detected by fluorescent microscopy or by flow cytometry

  12. MEASURING LYMPHOCYTE ACTIVITYFor detection of immunodeficiencies affecting T and/or B cell functions The activation of lymphocytes by a specific antigen is hardly detectable (low numbers of the antigen specific cells) The activation of lymphocytes by a polyclonal activator can help investigate abnormal lymphocyte functions

  13. POLYCLONAL ACTIVATION OF B AND T CELLS • Lectins (like concavalin A and PHA) act through crosslinking receptors • Intracellular signaling cascade activators (PMA – PKC activator, Ionomycin – increased intracellular Ca2+ levels) • Specific antibodies (anti-IgM, anti-CD3, anti-TCR)

  14. POLYCLONAL B CELL ACTIVATORS Phytolacca americana Pokeweed mitogen (PWM) Staphylococcus protein Asuperantigen (SpA) Epstein Bar Virus (EBV) (transforming) Anti-IgM antibody • Canavalia ensiformis • POLYCLONAL T CELL ACTIVATORS Phytohaemagglutinin(PHA) Concanavalin A (ConA) Anti-CD3, Anti-TCR antibodies Phaseolus vulgaris

  15. Receptor crosslinking (immediate) phosphorylation steps (seconds-minutes) • Western blot Antigen receptors (TCR, BCR), cytokine receptors, etc. • flow cytometry • fluorescent microscopy i.c. Ca2+ increase • qRT-PCR  mRNA • Western blot  protein Gene activation • i.c. cytometry Cytokine synthesis • ELISA • ELISPOT Cytokine secretion Lymphocyte activation The examination often requires specific Ag-Ab reactions Viability/apoptosis • dies specific to dead cells • 3H-thymidine • CFSE • MTT Cell division

  16. Fluo-3 or Indo-1 Fluorescence proportional with Intracellular Ca2+level activation of cells time basic signal MEASUREMENT OF CA2+ SIGNAL BY FLOW CYTOMETRY An increase in cytoplasmic Ca2+ levels can be detected by fluorescent indicator dyes /Fluo-3 or Indo-1/

  17. INTRACELLULAR CYTOKINE DETECTION BY IMMUNOFLUORESCENCE cytokine specific antibodies with fluorescent labeling • the cell membrane should be permeabilized (detergent) • but first the cells should be fixed to avoid decomposition (using e.g. aldehyde fixation) • optionally the cells can also be labeled by cell type (CD marker) specific antibodies cytokines

  18. INVESTIGATION OF GENE ACTIVATION Activation of cells can be monitored by the detection of mRNA transcription of the activated genes e.g. activation of cytokine genes QUANTITATIVE (REAL-TIME) PCR (qPCR/qRT-PCR) • cells  RNA isolation • RNA  reverse transcription (RT-PCR)  cDNA • cDNA  polymerase chain reaction (PCR) determination of quantity (investigation of gene activation on protein level  WB) the more mRNA the sample contains, the less time (cycles) it will take to reach the threshold

  19. ELISPOTEnzyme Linked Immuno-Spot • Similar principles as in ELISA • Determination of the number of cells that produce Ig, cytokines, chemokines, granzymes and other soluble effector molecules • Sensitive. Allows the determination of 1 activated cell among 300,000 others. (Can reveal activated effector cells not only after polyclonal but after antigen specific activation).

  20. ELISPOTEnzyme Linked Immuno-Spot - coating with antigen specific capture antibodies A spot showing the place of the cytokine producing cell - blocking - administration of the cells (activation, incubation) - washing - administration of biotin conjugated antigen-specific secondary antibody - avidin-enzyme conjugate - administration of the insoluble chromogenic substrate (AEC 3-amino-9-ethylcarbazol) Upper view of a well on an ELISPOT plate with the generated spots

  21. ELISPOTEnzyme Linked Immuno-Spot Spot number and size determination is valuated slowly and manually by microscopy or using “ELISPOT plate reader” which is fast and standardizable

  22. VIABILITY ASSAYS MTT (Dimethyl thiazolyldiphenyltetrazolium salt) Colorimetric test for measuring viability (apoptotic cells). NADPH-dependent cellular oxyreductaseenzymes that reduce MTT dye to an insoluble purple color (formazan). PI (propidium iodide) A fluorescent molecule intercalating with nucleic acids for measuring cell viability by flow cytometry. It is impermeable to viable cells. 7-AAD (7-aminoactinomycin) A fluorescent chemical intercalating with dsDNA. Won’t pass intact cells so is used for cell viability by flow cytometry.

  23. PROLIFERATION ASSAYS 3H-labeledthymidine- measures the increasing DNA content by β decomposition, and does not answer the numbers of cell division, and the dividing cell number. Bromodeoxyuridin (BrdU) A Thymidine-analogue can be administered to experimental animals, or cell cultures, and the proliferating cells can be detected by labelling with BrdU specific antibody (microscopy, FACS). CFSE (Carboxyfluoresceinsuccinimidyl ester) A fluorescent dye easily penetrating cells binding intracellular amine structures for long periods. Studies of cell divisions, prolifearation, migration and positioning.

  24. CFSETRACKING THE CELL DIVISIONS • „Cell tracer” dye enters the cell, and becomes trapped there • The apolar CFSE can bind covalently to the cellular proteins • Progressively halved within daughter cells • Used in vitro and in vivo to monitor lymphocyte proliferation CFSE-labeled cells that were not treated with polyclonal activator (control)

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