Cell Sorting

Flow Cytometry

Rob Salomonr.salomon@garvan.org.au

Why do we need Cell sorting ?

Heterogeneous samples provide problems for researcher 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 interest

History of droplet Cell Sorting

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)

Droplet Creation

Aria – 100um

SettingsAmpl = 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

Full Stream Overview

Influx 100um stream Amplitude ≈ 3.5Frequency = 39.00Pressure =24 PSI

Full Stream Overview

Full Stream Overview

Full Stream Overview

Full Stream Overview

Full Stream Overview

Last drop before break off

First break off drop

Satellite drop

Full Stream Overview

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

+++

---

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

Key Criteria for a Successful Sort

Instrument setup

Sample Preparation

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

Sort Masks

Yield Sort more drops

PurityAborts drop sort

Phase Aborts drop sort

BD FACSAria Users Guide

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

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

Useful Details