Flow cytometry is a powerful analytical tool that can analyze up to 10,000 individual cells per second. It works by passing single cells in suspension through a flow cell where they are exposed to a laser. Light scattering and fluorescence emissions from stained cells are then measured using detectors. Flow cytometry has many applications including analyzing cell viability, cell cycle, apoptosis, and cell sorting. It provides benefits like rapid analysis of single cells but also has limitations such as not showing intracellular protein localization and requiring cell preparation.

1. Flow Cytometry Anna Merca - Philippe Melas - Tolga Sütlü Stockholm Research School in Molecular Life Sciences 14 October 2005

2. Outline Introduction How Does Flow Cytometry Work? Applications Focus on Analysing Apoptosis Cell Sorting Benefits & Limitations

3. Introduction Powerful analytical tool up to 10000 individual cells/sec many properties at the same time wide area of application

4. How does it work? Cells in suspension (usually stained) 5 basic units Flow cell Light source Optical filter units Photomultiplier tubes Data processing and operating unit (software)

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. 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. Analysis Applications Viability and physiological state Cell cycle analysis & nucleic acid content Cell growth & death rates Intracellular calcium concentration Apoptosis Biotechnological Applications Bacterial Cultivations Yeast Cultivations Mammalian Cell Cultivations

8. Analyzing the graph -one color- CD3 NUMBER CD4 NUMBER CD8 NUMBER

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. Apoptosis vs. Necrosis Suicide Genetically programmed Cells shrink Condensation of chromatin Internucleosomal degradation of DNA Membrane retains its integrity MMP reduced Murder Random Event Cells swell Non-specific loss of chromatin structure Non-specific degradation of DNA Membrane loses its integrity

11. Apoptosis vs. Necrosis

12. How to study apoptosis: Caspases Fact : Caspase 3 is activated in apoptosis

13. How to study apoptosis: Mitochondrial Membrane Potential Fact: MMP is reduced during apoptosis

14. How to study apoptosis: Plasma Membrane Fact: Phosphatidyl serine (PS) flips from inside to the outside of the membrane during apoptosis

15. Cell Sorting 1 sample nozzle is vibrated sample stream breaks up into regular droplets droplets are electrostatically charged prior to passing through the laser 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

16. Cell Sorting 2 Charged Plates Collection Tubes

17. Benefits of Flow Cytometry measurement of single cells, identification of sub-populations wide area of application for analysis & diagnosis efficient and fast may be controlled remotely (online)

18. Limitations can not tell the intracellular location and distribution of proteins aggregates or debris can give false results pre-treatment of the cells for fluorescent staining is time-consuming samples such as tissue or cells in culture have to be treated to separate cells expensive and needs highly-trained technicians

19. Thanks for listening...