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.

1. Cell Sorting
Rob Salomon
r.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
Cytometry
Mechanical
•Uses a mechanical arm to catch cells of
interest
Electrostatic/ droplet
•Electrically charges droplets containing the
cells 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 Coulter
modifies the finishes, NIH introduced – replaced releases Epics
coulter funding begins mercury lamp (1972) (1977/8)
counter to (1969)
allow sorting
(1965)

7. Droplet Creation
Aria – 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 process
1. 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 Magic
but a good sort outcome doesn’t happen
automatically

25. Key Criteria for a Successful Sort
Instrument setup
Sample Preparation

26. Key Criteria for a Successful Sort
Instrument setup
• Nozzle choice
• Laser alignment and delay
• Drop delay - (set in drops but is actually a time figure)
• Collection vessel targeting
• Sort masks
Sample Preparation

27. Sort Masks
Yield
Purity
Phase
BD FACSAria Users Guide

28. 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
• 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