Flow cytometry training garvan

MLC Flow Cytometry Facility

Introduction To Flow
Cytometry
Basic training
Rob Salomon
Garvan Institute of Medical Research
Darlinghurst NSW

Flow Cytometry

What Is Flow Cytometry ?
Measurement

METRY

Cells Measurement

CYTO METRY

Flow Cells Measurement

Flow CYTO METRY

Flow Cells Measurement

Flow CYTO METRY

Flow Cytometry

Why use Flow Cytometry ?
• Rapid analysis ( 3k- 200k events/second)

• Individual event analysis

• Quantifiable results

• Multiple parameter analysis

• Statistical relevance

Why use Flow Cytometry
• Statistical Relevance

– As we increase our number of
observations we also increase
the ability to resolve smaller and
smaller changes The smallest flow
file will generally
contain at least
5000 events. It is
not unusual to
obtain >10^6 events

Flow and Imaging Comparison
Imaging Flow Cytometry

Cells per field/sec) Approx 100 20, 000

No. of parameter <6 <24
Quantifiable Maybe (using complex Yes
analysis tool) 18 bit resolution
12- 16 bit (< 65,536 (262,144 channels)
channels)
Ave number of 1,000 - >10, 000
analysed cells 10 field of 100
Anatomical localisation Yes no

Prerequisites for Flow
Cytometry

1. Cells in single cell suspension
2. Fluorescent probes
3. Cytometer

The key to good
result is good
sample
Preparation
http://www.photobiology.info/Zimmer.html -
from Roger Y.Tsien)

What does a Flow Cytometer
do?
Analyses light signals to determine:

Phenotype and Function

Cd3

Basics uses of Flow
Cytometry ?
• Phenotyping
• Apoptosis and cell death
• Cell cycle, cell divising and DNA synthesis
• Transduction/transfection confirmation
• Cell tracking
• Small particle analysis
• Functional analysis – calcium flux, gene expression, dye
efflux, mitochondrial activity

• Marine and microorganism identification

Flow Lab rules
• No access without training
• No unfixed human samples
• No unfixed PC2 samples
• Clean instrument after run
• Top up Sheath and empty waste at end
of run (waste has 100 ml bleach added to empty
container after emptying)

Instruments available
Analysers
– Calibur ( 2 Laser)
– Canto I (2 Laser)
– Canto II (3 Laser)
– LSRII CFI (5 Laser)
– LSRII SORP (7 Laser)

Sorters
– Aria
Operator assisted
– InFlux

Calibur*

2 laser
488nm ( blue)
633nm (red)

8 parameters
SSC & FSC
3 x blue laser
1 x red laser

Event rate
< 3, 000

Canto I
2 laser
488 nm ( blue)
633 nm (red)

8 parameters
SSC & FSC
4 x blue laser
2 x red laser

Event rate
< 15, 000

Canto II
3 laser
488nm ( blue)
633 nm (red)
405nm (violet)

10 parameters
SSC & FSC
4 x blue laser
2 x red laser
2 x violet

Event rate
< 10, 000

LSRII CFI 5 laser
488nm ( blue)
633 nm (red)
405nm (violet)
355nm (UV)
561 nm (YG)

10 parameters
SSC & FSC
4 x blue laser
2 x red laser
2 x violet

Event rate < 20, 000

7 laser
LSRII SORP ** 488nm ( blue)
633 nm (red)
405nm (violet)
355nm (UV)
561 nm (YG)
532nm (G)
514nm (514)

22 parameters
SSC & FSC& FSC
PMT
4 x blue
3 x red
4 x violet
2 UV
2 YG
3G
2 514

What’s inside a Flow
Cytometer ?
• Flow cytometers have 3 key systems

– Fluidics
– Optics
– Electronics

Fluidics system

Wet Sheath Flow
Waste
cart filter Cell

1. Top up at
start of run

Fluidics system

Wet Sheath Flow
Waste
cart filter Cell

1. Top up at start 2. Check and
of run remove air
bubbles

Fluidics system

Wet Sheath Flow
Waste
cart filter Cell

1. Top up at start 2. Check and 3. Ensure flow
of run remove air cell is free from
bubbles air and
blockages

Fluidics system

Wet Sheath Flow
Waste
cart filter Cell

1. Top up at start 2. Check and 3. Ensure flow 4. Ensure no air
of run remove air cell is free from bubbles in line
bubbles air and blockages and waste height
doesn’t change

Fluidics Prime
Canto I, and Canto II
1. Turn system on
2. Remove air From Sheath Filter
3. Perform software fluidics startup

Fluidics Prime
Calibur
1. Turn system on
3. Pressurise sheath tank
4. Prime 2 x ( no Tube)
5. Run TDw for 1 min – or until no air in waste
lines

Fluidics Prime
LSRII / LSRII SORP
1. Turn system on
3. Turn laser off (if possible)
4. Prime 2 x ( no Tube)
5. Run TDW for 1 min – or until no air in waste
lines

Flow Cell
Hydrodynamic
Low focusing of
sample to laser
intercept -
(interrogation point)

Medium

Legend
Laser intercept

High Core Stream

Optics

Sample laser
Laser Emission Spectral
Laser emission interaction at
delivery collection separation
flow cell

Ensure
lasers are on
- software
LSRII SORP
OR
Hardware

Optics

Sample laser
flow cell

Ensure
Ensure Clean Flow
lasers are on
cell
- software
LSRII SORP
OR
Hardware

Optics

Sample laser
flow cell

Ensure
Ensure Clean Flow Achieved by filter
lasers are on
Cell selection
- software
LSRII SORP
OR
Hardware

Optics
• Allows the excitation and the collection
of the emitted light
Steering
LASER mirrors

emission

Flow Cell -
Steering interrogation
mirrors
point

Optics cont..
Fluorescent
and SSC
Detectors

Signal Detection
FSC is achieved by
detector collecting emitted
or scattered light

Optics cont..
B530 Detector
– FITC GFP
530/30

488/10
SSC
Fluorescent and
Detector
506 LP SSC signals are
collected at right
Emission angles to the
from blue 575/26
excitation laser
laser are progressively
B575 Detector
picked off to
– PE, PI
556 LP
facilitate multiple
fluorochrome use

Spectral Separation
• Dichroic mirrors
• LP (Long Pass) – allows light longer than nominated
wavelength to pass
• SP (short Pass) – allows light shorter than nominated
wavelength to pass

• Band Pass filters
• Restrict the wavelength of light that is allowed to pass

Spectral Separation
• Band Pass filters
• Restrict the wavelength of
light that is allowed to pass

Centre of Width of
bandpass bandpass

Using PMT arrays
Channel Common
fluorochro
me
B 780 PE CY7

B 670 PE CY5
PerCP
B610 Dichroic
only
B575 PE

B 530 FITC/ GFP

488/10 SSC

Understanding PMT arrays

Dichroic ring

Band Pass ring
PMT ring

positi Wave
on length

A

B

C

D

E

F

A

positi Wave
on length

A >488nm

B

C

D

E

F

A

positi Wave
on length

A >488nm
B B

C

D

E

F

A

positi Wave
on length

A >488nm
B B >735nm

C

D

E

F

A

positi Wave
on length

C A >488nm

B B >735nm

C

D

E

F

A

positi Wave
on length

C A >488nm

B B >735nm

C 750-
810nm
D

E

F
A

pos Wave length
itio
n

C A >488nm

B B >735nm

C 750-810nm

D

E
D
F

A

pos Wave length
itio
n

C A >488nm

B B >735nm

C 750-810nm

D 488-735nm

E
D
F

A

pos Wave length
itio
n

C A >488nm
E
B B >735nm

C 750-810nm

D 488-735nm

E
D
F

A

pos Wave length
itio
n

C A >488nm
E
B B >735nm

C 750-810nm

D 488-735nm

E 655-735nm
D
F

A

pos Wave length
itio
n

E C A >488nm

B B >735nm
F C 750-810nm

D 488-735nm

E 655-735nm
D
F

A

pos Wave length
itio
n

E C A >488nm

B B >735nm
F C 750-810nm

D 488-735nm

E 655-735nm
D
F 670-735nm

A

Understanding the PMT
electronic
signal
Detector or PMT

Electron Cascade
Digitisation
Light and
signal processing

Amplification Voltage

http://sales.hamamatsu.com
/assets/applications/ETD/p
mt_handbook_complete.pdf

Affect of PMT voltage

Low voltage

Negative
population


Low voltage Mid Voltage

Negative Negative
population population


Low voltage Mid Voltage High Voltage

Negative Negative Negative
population population population

Types of signal
• Scatter light
• FSC and SSC
• Always the same wavelength as excitation
source
• Fluorescent light
• Always longer than the excitation source

Understanding Scatter Signals
• WBC discrimination

FSC has some
similarities to size

SSC has some
similarities to
granularity and
complexity

Fluorescent Signals
• Fluorescence may be used in the
detection of :
– Protein, RNA and DNA
– DNA synthesis
– Dye efflux
– Organelle Activity
A cytometer can
– Change in pH detected light from
– Protein interactions any system you
can design that
– Cell movement and division
utilises
– etc fluorescence

Examples of fluorescent probe
use

Understanding Fluoroscence

The fluorescent
Excited
molecule is excited
e-
by the excitation
state

e- source (laser). This
imparts energy to
e-
electrons in the
e-
molecule which in
Resting e-
then released as
Mechanism of the molecule
relaxes. The
Fluorscence energy is released
as light.

How do I choose my
Fluorochromes ?
• Antibody availability
• Function – i.e. Mcherry Vs GFP
• Fluorochrome brightness
• Excitation source
• Emission filters
• Other fluorochromes/ Signals present in my
sample (spectral overlap)

Fluorochrome Brightness
Probe QY
AF488 0.92
R-Pe 0.82
AF546 0.79
AF594 0.66
Quantum yield :
APC 0.68
Is a measure of the
A647 0.33 relative brightness of
eGFP 0.6 the fluorochrome. IT
is measured as:
Azumi Green 0.74
ZS Green 1 0.91

http://en.wikipedia.org/wiki/Fluorophore

Fluorescent protein table

http://www.tsienlab.ucsd.edu/Publications/Shaner%202005%20Nature%20Method
s%20-%20Choosing%20fluorescent%20proteins.pdf

Choosing your Fluorochromes

spectral
viewers
http://www.bdbioscience
s.com/research/multicolo
r/spectrum_viewer/index.
jsp

http://www.invitrogen.co
m/site/us/en/home/supp
ort/Research-
Tools/Fluorescence-
SpectraViewer.htmlUse
the

Understanding Spectral
Overlap
Effect of spectral overlap - Instrument View
120%

100%
Percentage of Signal

80%
in Detector

60%

40%

20%
Spectral overlap
0%
B 530 B 585 occurs when
PE 5% 87% fluorochromes
FITC 95% 13%
excited by the
same lasers emit
in similar ranges.

Compensation
Signal from FITC bright
Compensation Controls 120

Signal Strength
100
120% 80
60
100% 40
20
80%
Axis Title

0
overlap B 530 B 585
60%
FITC bright 100 13
40% overlap

20% FITC dull
0% 120
Signal Strength

B 530 B 585 100
FITC 100% 13% 80 Compensation is
PE 5% 100% 60
40 applied at the
20
0 single event
B 530 B 585
FITC dull 50 6 level

Effect of Compensation

Digital
compensation
doesn’t change the
underlying data it
just allows us to
Uncompensated Data interpret it

Effect of Compensation

Digital
compensation
doesn’t change the
underlying data it
just allows us to
Compensated Data interpret it

How many Fluorochromes can
I use ?
• Most flow = 1- 3 fluorochromes
• Basic phenotyping panel = 6-8
fluorochromes
• Complicated panels = 11-12
flourochromes
• High end = 17 fluorochromes
Seventeen-colour flow cytometry: unravelling the immune system
Stephen P. Perfetto, Pratip K. Chattopadhyay & Mario Roederer

Impact of increasing Flourochromes

• Data get dramatically more complex

Parameters 2 3 4 8 12 18 22

Populations 22 23 24 28 212 218 222

Populations 4 8 16 256 4,096 262,144 4,194,304

With 3 12 24 48 768 12,288 786,432 12,582,912
scatter
populations

Number of populations – assuming each fluorochromes gives
rise to only a positive and negative population

How do I get more ?
Analysis

Cell Sorting
Sorting

See It

Sort It

Contact Details
• Rob Salomon
– r.salomon@garvan.org.au
– (02) 9295 8431

• Bookings (David + Lachlan)
– Flow@garvan.org.au
– (02) 9295 8432

• http://linkage.garvan.unsw.edu.au/Flow/index.html

Data Acquisition
• BD FACSDiVa interface

Flow cytometry training garvan

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