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Cell Sorting _Flow Cytometry

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An introduction to cell sorting by Flow Cytometry. Including a brief overview of the history of droplet based cell sorting as well as how the instruments work. Some information adressing the key …

An introduction to cell sorting by Flow Cytometry. Including a brief overview of the history of droplet based cell sorting as well as how the instruments work. Some information adressing the key criteria to achieving a good cell sort outcome is also included.

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  • 1. Cell SortingRob Salomonr.salomon@garvan.org.au Flow Cytometry
  • 2. Why do we need Cell sorting ? Heterogeneous samples provide problems for researcher as the presence of non target cells can affect results
  • 3. What is Cell sorting ?
  • 4. Methods of Cell Sorting• Panning• Magnetic bead selection• Laser Capture microdisection• Microfluidics• Flow Cytometry based cell Sorting
  • 5. Cell Sorting using Flow CytometryMechanical•Uses a mechanical arm to catch cells ofinterestElectrostatic/ droplet•Electrically charges droplets containing thecells of interest
  • 6. History of droplet Cell Sorting Mark Fulwyer and Van Dyller Dick Sweet Joins BD releases 1st begin using Herzenberg lab commercial cell fluorescence (1971) sorter (1973/4) detection (1967)Mark Fulwyer Nasa funding Argon Ion laser Beckman Coultermodifies the finishes, NIH introduced – replaced releases Epicscoulter funding begins mercury lamp (1972) (1977/8)counter to (1969)allow sorting(1965)
  • 7. Droplet CreationAria – 100um Settings Ampl = Amplitude (how hard the stream is being vibrated) Freq = Frequency (how many droplets are being created per second – in KHz) Drop 1 = Arbitrary point of first separated drop (measures in pixels but must be a known time offset from the trigger) Gap = distance between drops ( in Pixels) – used for stream monitoring
  • 8. Full Stream Overview Influx 100um stream Amplitude ≈ 3.5 Frequency = 39.00 Pressure =24 PSI
  • 9. Full Stream Overview
  • 10. Full Stream Overview
  • 11. Full Stream Overview
  • 12. Full Stream Overview
  • 13. Full Stream Overview Last drop before break off First break off drop Satellite drop
  • 14. Full Stream Overview
  • 15. Sort process1. Particle enters stream
  • 16. Sort process• Particle enters stream• Particle triggers detectors
  • 17. Sort process• Particle enters stream• Particle triggers lasers• Particle progresses down the stream
  • 18. Sort process• Particle enters stream• Particle triggers lasers• Particle progresses down the stream• Particle enters last drop before breakoff
  • 19. Sort process• Particle enters stream• Particle triggers lasers• Particle progresses down the stream• Particle enters last drop before breakoff• Stream is charged
  • 20. Sort process• Particle enters stream• Particle triggers lasers• Particle progresses down the stream• Particle enters last drop before breakoff• Stream is charged• Droplet containing target particle separates from stream and retains charge
  • 21. Sort process• Particle enters stream• Particle triggers lasers• Particle progresses down the stream• Particle enters last drop before breakoff• Stream is charged• Droplet containing target particle separates from stream and retains charge• Stream is earthed
  • 22. Sort process • Particle enters stream • Particle triggers lasers • Particle progresses down the stream • Particle enters last drop before breakoff • Stream is charged • Droplet containing target particle+ - separates from stream and+ - retains charge+ - • Stream is earthed • Charged droplet enters electric field and is deflected
  • 23. Sort process• Particle enters stream• Particle triggers lasers• Particle progresses down the stream• Particle enters last drop before breakoff• Stream is charged• Droplet containing target particle separates from stream and retains charge• Stream is earthed• Charged droplet enters electric field and is deflected• Particle collected
  • 24. Sort process Its NOT Magicbut a good sort outcome doesn’t happen automatically
  • 25. Key Criteria for a Successful SortInstrument setupSample Preparation
  • 26. Key Criteria for a Successful SortInstrument setup • Nozzle choice • Laser alignment and delay • Drop delay - (set in drops but is actually a time figure) • Collection vessel targeting • Sort masksSample Preparation
  • 27. Sort Masks Yield Purity Phase BD FACSAria Users Guide
  • 28. Key Criteria for a Successful SortInstrument setup • Laser alignment and delay • Nozzle choice • Drop delay • Collection vessel targeting • Sort masksSample Preparation • Sample must be single cell • Match the cell concentration to the instrument setup and cell type • Stains should be fully worked up prior to sorting • controls are important
  • 29. Tips for good purities• Ensure good sample prep• Always use multiple doublet discrimination gates• Poorly separated populations cause uncertainty in population discrimination• Try to include both positive and negative selection criteria• The more criteria for selection the better• Never sort on an unstable stream
  • 30. Useful Details