FlowCytometry Basics

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FlowCytometry Basics

  1. 1. Flow Cytometry Anna Merca - Philippe Melas - Tolga Sütlü Stockholm Research School in Molecular Life Sciences 14 October 2005
  2. 2. Outline <ul><li>Introduction </li></ul><ul><li>How Does Flow Cytometry Work? </li></ul><ul><li>Applications </li></ul><ul><li>Focus on Analysing Apoptosis </li></ul><ul><li>Cell Sorting </li></ul><ul><li>Benefits & Limitations </li></ul>
  3. 3. Introduction <ul><li>Powerful analytical tool </li></ul><ul><li>up to 10000 individual cells/sec </li></ul><ul><li>many properties at the same time </li></ul><ul><li>wide area of application </li></ul>
  4. 4. How does it work? <ul><li>Cells in suspension (usually stained) </li></ul><ul><li>5 basic units </li></ul><ul><ul><li>Flow cell </li></ul></ul><ul><ul><li>Light source </li></ul></ul><ul><ul><li>Optical filter units </li></ul></ul><ul><ul><li>Photomultiplier tubes </li></ul></ul><ul><ul><li>Data processing and operating unit (software) </li></ul></ul>
  5. 5. Direct beam stop Laser Light High angle scatter : (Side Scatter) Cell structure Low angle scatter : (Forward Scatter) Cell size Fluorescence at longer wavelengths Flow cell Direction of flow
  6. 6. Basic Optics of a Flow Cytometer ( An automated fluorescent microscope) Dichroic mirrors 1 2 3 Laser(s) Cell Collection Lenses Scatter Low & High angle Photomultiplier tubes
  7. 7. Analysis Applications <ul><li>Viability and physiological state </li></ul><ul><li>Cell cycle analysis & nucleic acid content </li></ul><ul><li>Cell growth & death rates </li></ul><ul><li>Intracellular calcium concentration </li></ul><ul><li>Apoptosis </li></ul><ul><li>Biotechnological Applications </li></ul><ul><ul><li>Bacterial Cultivations </li></ul></ul><ul><ul><li>Yeast Cultivations </li></ul></ul><ul><ul><li>Mammalian Cell Cultivations </li></ul></ul>
  8. 8. Analyzing the graph -one color- CD3 NUMBER CD4 NUMBER CD8 NUMBER
  9. 9. Analyzing the graph CD4 0 1 10 2 10 3 10 4 0 10 CD3 CD3 CD4 CD3+CD4+ green CD3-CD4+ cyan CD3+CD4- cyan CD3-CD4- black
  10. 10. Apoptosis vs. Necrosis <ul><li>Suicide </li></ul><ul><li>Genetically programmed </li></ul><ul><li>Cells shrink </li></ul><ul><li>Condensation of chromatin </li></ul><ul><li>Internucleosomal degradation of DNA </li></ul><ul><li>Membrane retains its integrity </li></ul><ul><li>MMP reduced </li></ul><ul><li>Murder </li></ul><ul><li>Random Event </li></ul><ul><li>Cells swell </li></ul><ul><li>Non-specific loss of chromatin structure </li></ul><ul><li>Non-specific degradation of DNA </li></ul><ul><li>Membrane loses its integrity </li></ul>
  11. 11. Apoptosis vs. Necrosis
  12. 12. How to study apoptosis: Caspases <ul><li>Fact : Caspase 3 is activated in apoptosis </li></ul>
  13. 13. How to study apoptosis: Mitochondrial Membrane Potential <ul><li>Fact: MMP is reduced during apoptosis </li></ul>
  14. 14. How to study apoptosis: Plasma Membrane <ul><li>Fact: Phosphatidyl serine (PS) flips from inside to the outside of the membrane during apoptosis </li></ul>
  15. 15. Cell Sorting 1 <ul><li>sample nozzle is vibrated </li></ul><ul><li>sample stream breaks up into regular droplets </li></ul><ul><li>droplets are electrostatically charged prior to passing through the laser </li></ul><ul><li>droplets containing cells of interest (as characterized by scatter or fluorescence properties) are deflected into tubes by passing them through a pair of charged plates </li></ul>
  16. 16. Cell Sorting 2 Charged Plates Collection Tubes
  17. 17. Benefits of Flow Cytometry <ul><ul><li>measurement of single cells, identification of sub-populations </li></ul></ul><ul><ul><li>wide area of application for analysis & diagnosis </li></ul></ul><ul><ul><li>efficient and fast </li></ul></ul><ul><ul><li>may be controlled remotely (online) </li></ul></ul>
  18. 18. Limitations <ul><li>can not tell the intracellular location and distribution of proteins </li></ul><ul><li>aggregates or debris can give false results </li></ul><ul><li>pre-treatment of the cells for fluorescent staining is time-consuming </li></ul><ul><li>samples such as tissue or cells in culture have to be treated to separate cells </li></ul><ul><li>expensive and needs highly-trained technicians </li></ul>
  19. 19. Thanks for listening...

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