Aims cell sorting talk 1

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Cell Sorting talk given by Rob Salomon to AIMS Haem Discussion Group Meeting. Monday 4 November 630 pm, Liverpool Hospital, Pathology Tutorial Room, 2nd Floor Can you talk on "An Introduction to Cell Sorting"

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Aims cell sorting talk 1

  1. 1. Robert Salomon Flow Cytometry Manager/ Senior Scientist www.flow.garvan.org.au 9295 8432 office 9295 8431 lab
  2. 2. Why do we need Cell sorting ? Heterogeneous samples provide problems for researchers as the presence of non target cells can affect results
  3. 3. What is Cell sorting ?
  4. 4. Methods of Cell Sorting • • • • • Panning Magnetic bead selection Laser Capture microdisection Microfluidics Flow Cytometry based cell Sorting
  5. 5. Cell Sorting using Flow Cytometry Mechanical • Uses a mechanical arm to catch cells of interest Electrostatic/ Droplet • Electrically charges droplets containing the cells of interest Microfluidics
  6. 6. History of droplet Cell Sorting Mark Fulwyer and Van Dyller begin using fluorescence detection (1967) 1965 Mark Fulwyer modifies the coulter counter to allow sorting (1965) BD releases 1st commercial cell sorter (1973/4) Dick Sweet Joins Herzenberg lab (1971) 1969 Nasa funding finishes, NIH funding begins (1969) 1971 1972 Argon Ion laser introduced – replaced mercury lamp (1972) 1973/4 1977/8 Beckman Coulter releases Epics (1977/8)
  7. 7. Full Stream Overview Nozzle Orrifice Laser intercept/ Signal Generation Droplet propagation Droplet breakoff Droplet defelection
  8. 8. Full Stream Overview Nozzle Orifice
  9. 9. Full Stream Overview Laser Intercept
  10. 10. Full Stream Overview Signal Generation
  11. 11. Full Stream Overview Droplet Breakoff Last drop before break off First break off drop Satellite drop
  12. 12. Full Stream Overview Droplet Deflection
  13. 13. Droplet Creation- the heart of electrostatic cell sorting Last drop before breakoff Breakoff point Instrument Controls Amplitude = how hard the stream is being vibrated- Defines the distance from nozzle to first drop Frequency – defines the number First drop after breakoff Satellite drop Merged Satellite of droplets being created per second (usually in the 5- 90 KHz range) Pressure = user defined – combined with frequency allows user to generate stable droplet formation
  14. 14. Sort process 1. Particle enters stream
  15. 15. Sort process 1. Particle enters stream 2. Particle triggers detectors
  16. 16. Sort process 1. Particle enters stream 2. Particle triggers lasers 3. Particle progresses down the stream
  17. 17. Sort process 1. 2. 3. 4. Particle enters stream Particle triggers lasers Particle progresses down the stream Particle enters last drop before breakoff
  18. 18. Sort process 1. 2. 3. 4. Particle enters stream Particle triggers lasers Particle progresses down the stream Particle enters last drop before breakoff 5. Stream is charged
  19. 19. Sort process 1. 2. 3. 4. Particle enters stream Particle triggers lasers Particle progresses down the stream Particle enters last drop before breakoff 5. Stream is charged 6. Droplet containing target particle separates from stream and retains charge
  20. 20. Sort process 1. 2. 3. 4. Particle enters stream Particle triggers lasers Particle progresses down the stream Particle enters last drop before breakoff 5. Stream is charged 6. Droplet containing target particle separates from stream and retains charge 7. Stream is earthed
  21. 21. Sort process 1. 2. 3. 4. 5. 6. + + + - 7. 8. 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
  22. 22. Sort process 1. 2. 3. 4. 5. 6. 7. 8. 9. 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
  23. 23. Sort process Cell Sorting Its NOT Magic but a good sort outcome doesn’t happen automatically
  24. 24. Key Criteria for a Successful Sort Instrument setup Sample Preparation
  25. 25. Key Criteria for a Successful Sort Instrument setup • Nozzle choice – big cells require a bigger nozzle and sorting is slower • Good stable Laser alignment and delay – basic analysis requirement • Drop delay – droplet breakoff needs to remain stable otherwise you will sort the wrong drop • Collection vessel targeting - especially true for plate sorting • Sort masks - defines purity, recovery and accuracy of stream trajectory Sample Preparation
  26. 26. Key Criteria for a Successful Sort Instrument setup • • • • • Laser alignment and delay Nozzle choice Drop delay Collection vessel targeting Sort masks Sample Preparation • Sample must be single cell – sorting doublet gives poor purities • Match the cell concentration to the instrument setup and cell type • Controls are important – Analysis must be correct at time of sorting
  27. 27. 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 • Never sort on an unstable Instrument
  28. 28. Useful Details
  29. 29. Uses for cell Sorting
  30. 30. Analysis Uses for cell Sorting Sorting Cell Sorting See Sort It It
  31. 31. Contact Details Rob Salomon r.salomon@garvan.org.au flow@garvan.org.au 9295 8432 office 9295 8431 lab www.flow.garvan.org.au This presentation: http://www.slideshare.net/Robert_Salomon/aimscell-sorting-talk-1

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