Robert SalomonFlow Cytometry Manager/ Senior Scientist

www.flow.garvan.org.au9295 8432 office 9295 8431 lab

Why do we need Cell sorting ?

Heterogeneous samples provide problems for researchers as the presence of non target cells can affect results

What is Cell sorting ?

Methods of Cell Sorting

• Panning • Magnetic bead selection• Laser Capture microdisection• Microfluidics• Flow Cytometry based cell Sorting

Cell Sorting using Flow Cytometry

Mechanical • Uses a mechanical arm to catch cells of

interest Electrostatic/ Droplet• Electrically charges droplets containing the

cells of interestMicrofluidics

History of droplet Cell Sorting

1965 1969 1971 1972 1973/4 1977/8

Mark Fulwyer modifies the coulter counter to allow sorting (1965)

Mark Fulwyer and Van Dyller begin using fluorescence detection (1967)

BD releases 1st commercial cell sorter (1973/4)

Beckman Coulter releases Epics (1977/8)

Argon Ion laser introduced – replaced mercury lamp (1972)

Dick Sweet Joins Herzenberg lab (1971)

Nasa funding finishes, NIH funding begins (1969)

Full Stream Overview

Nozzle Orrifice

Laser intercept/ Signal Generation

Droplet propagation

Droplet breakoff

Droplet defelection

Full Stream Overview

Nozzle Orifice

Full Stream Overview

Laser Intercept

Full Stream Overview

Signal Generation

Full Stream Overview

Last drop before break off

First break off drop

Satellite drop

Droplet Breakoff

Full Stream Overview

Droplet Deflection

Droplet Creation- the heart of electrostatic cell sorting

Instrument ControlsAmplitude = how hard the stream is being vibrated- Defines the distance from nozzle to first drop

Frequency – defines the number 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

Last drop before breakoff

First drop after breakoff

Satellite drop

Merged Satellite

Breakoff point

Sort process 1. Particle enters stream

Sort process 1. Particle enters stream2. Particle triggers detectors

Sort process 1. Particle enters stream2. Particle triggers lasers3. Particle progresses down the stream

Sort process 1. Particle enters stream2. Particle triggers lasers3. Particle progresses down the stream 4. Particle enters last drop before

breakoff

Sort process 1. Particle enters stream2. Particle triggers lasers3. Particle progresses down the stream 4. Particle enters last drop before

breakoff 5. Stream is charged

Sort process 1. Particle enters stream2. Particle triggers lasers3. Particle progresses down the stream 4. Particle enters last drop before

breakoff 5. Stream is charged6. Droplet containing target particle

separates from stream and retains charge

Sort process 1. Particle enters stream2. Particle triggers lasers3. Particle progresses down the stream 4. Particle enters last drop before

breakoff 5. Stream is charged6. Droplet containing target particle

separates from stream and retains charge

7. Stream is earthed

Sort process 1. Particle enters stream2. Particle triggers lasers3. Particle progresses down the stream 4. Particle enters last drop before

breakoff 5. Stream is charged6. Droplet containing target particle

separates from stream and retains charge

7. Stream is earthed8. Charged droplet enters electric field

and is deflected

+++

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Sort process 1. Particle enters stream2. Particle triggers lasers3. Particle progresses down the stream 4. Particle enters last drop before

breakoff 5. Stream is charged6. Droplet containing target particle

separates from stream and retains charge

7. Stream is earthed8. Charged droplet enters electric field

and is deflected 9. Particle collected

Sort process

Its NOT Magic

but a good sort outcome doesn’t happen automatically

Cell Sorting

Key Criteria for a Successful Sort

Instrument setup

Sample Preparation

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

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

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

Useful Details

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Uses for cell Sorting

Uses for cell Sorting

Cell SortingSee It

Sort It

Analy

sis

Sort

ing

Contact Details

Rob Salomonr.salomon@garvan.org.auflow@garvan.org.au9295 8432 office 9295 8431 labwww.flow.garvan.org.au

This presentation: http://www.slideshare.net/Robert_Salomon/aims-cell-sorting-talk-1