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"
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
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
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
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