Flow Cytometry Training: Introduction day 1 session 2

Day 1
Introduction
10:30 To
1:00 pm
Session 2
FLOW CYTOMETRY
TRAINING
Robert Salomon
(Flow Manager and Senior Flow Cytomerty Scientist)

Theory Session – 0900 till 1300
 Introductions to the Lab and Staff
 Self Introductions
 Basics of flow cytometry
 Applications for Flow Cytometry
Morning tea - Provided
 Getting Started
 Panel Design
 Controls and compensation
 Data Analysis and Interpretation
 Data Acquisition Overview
 Instrumentation
Lunch – provided
Practical Session
SESSION 2
Outline – 5 mins
Day 1

 To design good Flow Cytometry Experiments you’ll need to
 Start with a good panel
 Use appropriate controls
 Compensation controls
 Fluorescence minus one (FMO)controls
 Positive and negative controls
 Monitor your instrument
GETTING STARTED

 What is a “PANEL” ?
 A Panel is a combination of fluorochromes that allows us to
characterise our sample using flow cytometry.
 It requires balancing technical and biological factors so that we can
accurately interpret the biological state of our cells
PANEL DESIGN

 Understanding Fluorochromes
PANEL DESIGN
Input Light
Energy
Output Light
Energy
Fluorochrome

 Getting started on your panel
 Know your Sample and Desired outcome
 What is the goal of the experiment ?
 Which markers are critical ?
 Refer to literature for abundance & co-expression of antigen
 Know your Fluorochormes and Instruments
 Fluorochrome brightness
 Spectral overlap
 What excites the fluor and what is the emmision
 Theoretically design an “optimal Panel”
 Optimise the individual Elements in the Panel
 Antibody titration
 Put it all together : Test the “optimised Panel”
 Use all relevant controls
 Refine the panel
PANEL DESIGN

 What are the important
epitopes ?
 How abundant are they ?
 Which of my
characteristics of interest
are co-expressed ?
 Which characteristics are
negative for my cells of
interest ? (dead cell
exclusion dyes)
PANEL DESIGN:
KNOW YOUR SAMPLE

 Choice of Fluorchorome is critical , especially in more
complex panels >4 fluors
 What restrictions do I have ?
 Is there anything intrinsic in my sample that I will restrict my choices?
 What fluorochromes can I see on the instrument?
 What antibody/ reagents are available to me ?
 What is the best combination available?
 Can I match my least abundant epitope to the brightest fluorochrome?
 Who do my fluorochromes interact ?
PANEL DESIGN:
KNOW YOUR FLUOROCHROMES

 Is there anything intrinsic in my sample that I will restrict my
choices?
 Fluorescent Proteins
 Auto Fluorescence
PANEL DESIGN:
FLUOROCHROME RESTICTIONS

 What fluorochromes can I see on the instrument?
PANEL DESIGN:
http://flow.garvan.org.au/flow-
cytometers-instrument-details

 What fluorochromes antibody combinations are available?
PANEL DESIGN:

 Fluorochrome Brightness
1. Quantum Efficiency = How well
the fluor is excited.
1. Quantum Yield = How well the
fluor converts excitation into
emission.
PANEL DESIGN:
OPTIMISING FLUOROCHROME COMBINATIONS

 Stain Index gives a measure of how well the positive
population separares from the negative population
SI =(MFI pos – MFI neg )/2 x SD MFI neg pop
 Is a combination of:
1. Epitope expression level
2. Fluorochrome
3. Cell type
4. The pressence of other fluors in the sample
PANEL DESIGN:

PANEL DESIGN:
Epitope Expression Level
Fluorochrome Brightness

PANEL DESIGN:
THEORETICAL PANELS
Name Rob Salomon
Panel Name 2015 B/T Sep
Desired Outcome Separation of B cells from CD4 and CD8 Tcells
Instrument Canto II
PANEL 1
parameters target Expression level Fluor Fluor Brightness channel
1 Cd3 high GFP ++ B530
2 cd4 high PE ++++ B585
3 cd8 high APC ++++ R660
4 cd19 very low APC Cy7 + R780
5 Ter119 very high PE CY7 +++ B780
6 Death DAPI +++++ V450
7
8
PANEL 2
1 Cd3 high GFP ++ B530
2 cd4 high APC CY7 ++++ R780
4 cd19 very low PE ++++ B585
5 Ter119 very high Percp cy5.5 +++ B695
7
8

 Antibody titration
 To establish the optimum antibody dilution, highest signal/noise ratio
 Done for each antibody in the correct experimental condition
PANEL DESIGN:
OPTIMISE INDIVIDUAL ELEMENTS
http://regmed.musc.edu/flowcytometry/images/AntibodyTitration.jpg
Note: tandem dyes may
require lot-specific
titration

 Depending on type of assay.
 Determining changes in level of expression
 Absolutely requires saturating levels
 Differentiating between cell types.
 Can be achieved through non-saturating however care should be taken
PANEL DESIGN:
Saturating
4
7
4
5/7
Non
Saturating
01

 FMO (fluorescence minus one)
 Contains all markers except one
 To discriminate positive vs negative populations
PANEL DESIGN:
http://www.dartmouth.edu/~dartlab/?page=flow-cytometry

Name Rob Salomon
Panel Name 2015 B/T Sep
Desired Outcome Separation of B cells from CD4 and CD8 Tcells
Instrument Canto II
PANEL 2
1 cd3 high GFP ++ B530
2 cd4 high APC CY7 ++++ R780
4 cd19 very low PE ++++ B585
5 Ter119 very high Percp cy5.5 +++ B695
7
8
PUTTING IT ALL TOGETHER:
TEST YOUR COMPLETE PANEL
Panel Plus Controls and Analyse

Unstained control
As negative controls – no antibody present
To assess any autofluorescence
Compensation controls
Single colour controls – stain with one fluorophore
with the EXACT conditions as experimental samples
Compensate for fluorophore emission overlaps
CONTROLS:
INSTRUMENT & SETUP CONTROLS
http://www.abdserotec.com/flow-cytometry-fluorescence-compensation.html

Unstained controls
 Allow relative Determination of positivity
CONTROLS
Question : Which Populations is the Positive ?

Unstained controls
 Allow relative Determination of positivity
CONTROLS
Question : Which Populations is the Positive ?
Answer : Both – Because I spiked in a
negative control

 Compensation Controls
 Set of samples/beads consisting of
 One tube of unstained sample/beads +
 Tubes of single fluorochrome labelled samples
OR
 Tubes of single fluorochrome labelled beads spiked with unstained beads.
CONTROLS

EFFECT OF COMPENSATION
Digital
compensation
doesn’t change the
underlying data it
just allows us to
interpret it

DIGRESSION:
WHY DO WE NEED TO COMPENSATE ?
spectral
viewers
http://www.bdbiosci
ences.com/research
/multicolor/spectru
m_viewer/index.jsp
http://www.invitroge
n.com/site/us/en/h
ome/support/Resear
ch-
Tools/Fluorescence-
SpectraViewer.html

DIGRESSION:
spectral
viewers
http://www.bdbiosci
ences.com/research
/multicolor/spectru
m_viewer/index.jsp
http://www.invitroge
n.com/site/us/en/h
ome/support/Resear
ch-
Tools/Fluorescence-
SpectraViewer.htmlU
se the

DIGRESSION:
Spectral overlap
occurs when
fluorochromes
excited by the
same lasers
emit in similar
ranges.
B 530 B 585
PE 5% 87%
FITC 95% 13%
0%
20%
40%
60%
80%
100%
120%
PercentageofSignal
inDetector
Effect of spectral overlap - Instrument View

COMPENSATION THEORY
Compensation
is applied at
the single
event level
B 530 B 585
FITC 100% 13%
PE 5% 100%
0%
20%
40%
60%
80%
100%
120%
AxisTitle
Signal from Compensation
Controls
overlap
overlap
B 530 B 585
FITC bright 100 13
0
20
40
60
80
100
120
SignalStrength
FITC bright
B 530 B 585
FITC dull 50 6
0
20
40
60
80
100
120
SignalStrength
FITC dull

 Compensation removes the signal spillover from one
fluorochrome into any other parameter.
COMPENSATION THEORY
MFI pos population NTC
1 = MFI neg population NTC
1
2
………………………………..
n = MFI neg population NTC
n
MFI = Media Fluorescent Intensity
NTC = Non Target channel
MFI FITC
channel
MFI Pe
channel
MFI APC
channel
Fitc 25818 193 222
Pe 421 23940 228
APC 431 181 27271
unstained 905 377 235

 Compensation removes the signal spillover from one
fluorochrome into any other parameter.
COMPENSATION THEORY
1
2
………………………………..
n = MFI neg population NTC
n
MFI = Media Fluorescent Intensity
NTC = Non Target channel
Where
D= Fluorescence in detector
F= Fluorescence signal
N = FL from detector #
N = FL in Detector #

DATA ANALYSIS AND INTERPRETATION

PLOT TYPES
Dot Plot
Histogram Histogram
Contour Plot

 Rectangular Gates
 Elliptical Gates
 Polygon Gates
 Quadrant Gates
 Histogram regions
GATES & GATING
Rough gates – generally
suitable for initial gating
Generally better suited to
biological populations
Gives the most control –
generally recommended
Flow data generally doesn’t
conform to 900 angles
Only applicable for histograms

 Gating Hierarchy
HIERARCHY

 Gating Hierarchy
HIERARCHY
1
2
3
4
5a
5b

 Numbers
 Percentages – parent and total
 MFI - Median/Mean Fluorescent intensity
 CV’s
STATISTICS

 As we increase our number of observations we also increase the ability
to resolve smaller and smaller changes
STATISTICAL RELEVANCE IN FLOW
CYTOMETRY
The smallest flow file will
generally contain at least
5000 events. It is not unusual
to obtain >10^6 events.
 http://www.dako.com/08065_15dec05_guide_to_flow_cytometry.
pdf

 Data must be on scale
 There must be controls to show the relationship between
populations
 Instrument settings must be constant
 Be careful viewing uncompensated data
 Do not over interpret results (especially without the correct
controls)
HOW TO INTERPRET PLOTS

 Out of scale data cannot be read efficiently.
DATA MUST BE ON SCALE
Right most population off scale

1. Decide what your looking for
2. Decide on the logic used to identify the population of
interest
3. Label Everything
4. Draw your plots
5. Open you gate hierarchy
6. Draw the Gates
7. Chose your statistics of interest
CREATING AN ANALYSIS TEMPLATE

INSTRUMENTATION
Analysers
 FACSCalibur
 CantoI
 CantoII
 AMR Fortessa
 LSRII SORP
Sorters and
Cell Separation
 FACSAriaIIu
 FACSAriaIII (x 2)
 AutoMacs Pro

FACS CALIBUR*
2 laser
488nm (blue)
633nm (red)
8 parameters
SSc & FSc
blue laser - 3x
red laser - 1x
Event rate
< 3, 000

CANTO I
2 laser
488nm (blue)
633nm (red)
8 parameters
SSc & FSc
blue laser - 4x
red laser - 2x
Event rate
< 15, 000

CANTO II
3 laser
405nm (violet)
488nm (blue)
633nm (red)
10 parameters
SSc & FSc
violet - 2x
blue laser - 4x
red laser - 2x
Event rate
< 10, 000

AMR FORTESSA
4 laser
405nm (violet)
488nm (blue)
533nm (YG)
633nm (red)
15 parameters
SSC & FSC
violet - 4x
blue laser - 2x
yellowgreen - 4x
red laser - 3x
Event rate
< 20, 000

LSRII SORP **
5 laser
355nm (UV)
405nm (violet)
488nm (blue)
561nm (YG)
633nm (red)
20 parameters
SSc & FSc
& FSc PMT
UV - 2x
violet - 6x
blue - 2x
YG -4x
red - 3x
Event rate
< 20, 000

ARIA IIU CELL SORTER
3 laser
405nm (violet)
488nm (blue)
633nm (red)
12 parameters
SSc & FSc
violet - 2x
blue - 6x
red - 2x
Event rate
< 20, 000

ARIA III CELL SORTER
4 laser
405nm (violet)
488nm (blue)
561nm (YG)
633nm (red)
18 parameters
SSc & FSc
violet - 6x
blue - 2x
YG - 5x
red - 3x
Event rate
< 20, 000

AUTOMACS PRO
Magnetic Cell
Separation
Technique.
Speed ~4ml in
15mins

WHAT’S INSIDE A FLOW CYTOMETER ?
 Flow cytometers have 3 key systems
 Fluidics
 Optics
 Electronics

FLUIDICS SYSTEM
Wet
cart
Sheath
filter
Flow
Cell
Waste
1. Top up at start of
run

FLUIDICS SYSTEM
Wet
cart
Sheath
filter
Flow
Cell
Waste
1. Top up at start
of run
2. Check and
remove air
bubbles

FLUIDICS SYSTEM
Wet
cart
Sheath
filter
Flow
Cell
Waste
1. Top up at start
of run
2. Check and
remove air
bubbles
3. Ensure flow
cell is free from
air and
blockages

FLUIDICS SYSTEM
Wet
cart
Sheath
filter
Flow
Cell
Waste
1. Top up at start
of run
2. Check and
remove air
bubbles
3. Ensure flow cell
is free from air
and blockages
4. Ensure no air
bubbles in line
and waste height
doesn’t change

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

FLUIDICS PRIME
1. Turn system on
2. Remove air From Sheath Filter
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
6. Turn laser on.
Calibur LSRII / LSRII SORP

FLOW CELL
Low
Medium
High
Hydrodynamic
focusing of
sample to laser
intercept -
(interrogation point)
Legend
Laser intercept
Core Stream

FLOW CELL
Hydrodynamic
focusing of
sample to laser
intercept -
(interrogation point)
Low
Medium
High
Legend
Laser intercept
Core Stream
Laser focal
plane
Signal
spread

OPTICS
Laser emission
Laser
delivery
Sample laser
interaction at
flow cell
Emission
collection
Spectral
separation
Ensure lasers
are on -
software
LSRII SORP
OR
Hardware

OPTICS
Laser emission
Laser
delivery
Sample laser
interaction at
flow cell
Emission
collection
Spectral
separation
Ensure lasers
are on -
software
LSRII SORP
OR
Hardware
Ensure Clean Flow cell

OPTICS
Laser emission
Laser
delivery
Sample laser
interaction at
flow cell
Emission
collection
Spectral
separation
Ensure lasers
are on -
software
LSRII SORP
OR
Hardware
Ensure Clean Flow
Cell
Achieved by filter
selection

OPTICS
 Allows the excitation and the collection of the emitted light
LASER
Steering
mirrors
Steering
mirrors
Flow Cell -
interrogation
point
emission

OPTICS CONT..
Signal Detection
is achieved by
collecting
emitted or
scattered light
Forward Scatter (FSc)
detector
Fluorescent and Side
Scatter (SSc) Detectors

 Dichroic mirrors bounce light
 Bandpass filter clean up the signal
HOW DO WE COLLECT MULTIPLE SIGNALS
FROM THE ONE EXCITATION SOURCE ?
Dichroic
Mirror

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
bandpass
Width of
bandpass

UNDERSTANDING PMT ARRAYS
Dichroic ring
Band Pass ring
PMT ring

USING PMT ARRAYS
Channel Common
fluorochrome
B 780 PE CY7
B 670 PE CY5 PerCP
B 610 Dichroic only
B 575 PE
B 530 FITC/ GFP
488/10 SSC

Position Wave length
A
B
C
D
E
F
A

A >488nm
B
C
D
E
F
A

A >488nm
B
C
D
E
F
A
B

position Wave length
A >488nm
B >735nm
C
D
E
F
A
B

A >488nm
B >735nm
C
D
E
F
A
B
C

A >488nm
B >735nm
C 750-810nm
D
E
F
A
B
C

A >488nm
B >735nm
C 750-810nm
D
E
F
A
B
C
D

A >488nm
B >735nm
C 750-810nm
D 488-735nm
E
F
A
B
C
D

A >488nm
B >735nm
C 750-810nm
D 488-735nm
E
F
A
B
C
D
E

A >488nm
B >735nm
C 750-810nm
D 488-735nm
E 655-735nm
F
A
B
C
D
E

A >488nm
B >735nm
C 750-810nm
D 488-735nm
E 655-735nm
F
A
B
C
D
E
F

A >488nm
B >735nm
C 750-810nm
D 488-735nm
E 655-735nm
F 670-735nm
A
B
C
D
E
F

UNDERSTANDING THE PMT
Detector or PMT
Amplification
Voltage
Electron
Cascade Digitisatio
n and
processing
http://sales.hamamatsu.com/assets/applicati
ons/ETD/pmt_handbook_complete.pdf
Light
signal
electronic
signal

AFFECT OF PMT VOLTAGE
Low voltage
Negative
population

Mid Voltage
Negative
population
Negative
population
Low voltage

High Voltage
Negative
population ???
Negative
population ???
Negative
population ???
Mid VoltageLow voltage

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