Aims cell sorting talk 1
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The document discusses the necessity and methodologies of cell sorting, highlighting issues with heterogeneous samples and the various techniques available, particularly focusing on flow cytometry-based sorting. It details the technical components and processes involved in electrostatic droplet sorting, including key considerations for successful sorting outcomes like instrument setup and sample preparation. Additionally, it provides historical context and advancements in the field of cell sorting.
Aims cell sorting talk 1
- 1. Robert Salomon Flow Cytometry Manager/ Senior Scientist www.flow.garvan.org.au 9295 8432 office 9295 8431 lab
- 2. Why do we need Cell sorting ? Heterogeneous samples provide problems for researchers 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 Cytometry Mechanical • Uses a mechanical arm to catch cells of interest Electrostatic/ Droplet • Electrically charges droplets containing the cells of interest Microfluidics
- 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. Full Stream Overview Nozzle Orrifice Laser intercept/ Signal Generation Droplet propagation Droplet breakoff Droplet defelection
- 8. Full Stream Overview Nozzle Orifice
- 9. Full Stream Overview Laser Intercept
- 10. Full Stream Overview Signal Generation
- 11. Full Stream Overview Droplet Breakoff Last drop before break off First break off drop Satellite drop
- 12. Full Stream Overview Droplet Deflection
- 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. Sort process 1. Particle enters stream
- 15. Sort process 1. Particle enters stream 2. Particle triggers detectors
- 16. Sort process 1. Particle enters stream 2. Particle triggers lasers 3. Particle progresses down the stream
- 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. 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. 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. 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. 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. 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. Sort process Cell Sorting Its NOT Magic but a good sort outcome doesn’t happen automatically
- 24. Key Criteria for a Successful Sort Instrument setup Sample Preparation
- 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. 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. 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. Useful Details
- 29. Uses for cell Sorting
- 30. Analysis Uses for cell Sorting Sorting Cell Sorting See Sort It It
- 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