flow cytometry training: introduction day 1 session 2

Day 1Introduction 10:30 To 1:00 pm

Session 2

FLOW CYTOMETRY TRAINING

Robert Salomon (Flow Manager and Senior Flow Cytomerty Scientist)

Theory Sess ion – 0900 t i l l 1300

Introductions to the Lab and Staff Self Introductions Basics of flow cytometry Applications for Flow Cytometry

Morn ing tea - P rov ided

Getting Started Panel Des ign Controls and compensat ion Data Analys is and Interpretat ion

Data Acquisition Overview InstrumentationLunch – prov ided

Pract ica l Sess ion

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?


http://flow.garvan.org.au/flow-cytometers-instrument-details




What fluorochromes antibody combinations are available?


Fluorochrome Brightness

1. Quantum Efficiency = How well the fluor is excited.

2. 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 level2. Fluorochrome3. Cell type4. The pressence of other fluors in the sample

PANEL DESIGN:OPTIMISING FLUOROCHROME

COMBINATIONS

PANEL DESIGN:OPTIMISING FLUOROCHROME

COMBINATIONS

Epitope Expression Level

Fluorochrome Brightness

PANEL DESIGN:THEORETICAL PANELS

Name Rob SalomonPanel Name 2015 B/T Sep

Desired Outcome Separation of B cells from CD4 and CD8 Tcells

Instrument Canto II

PANEL 1parameters target Expression level Fluor Fluor Brightness channel

1 Cd3 high GFP ++ B5302 cd4 high PE ++++ B5853 cd8 high APC ++++ R6604 cd19 very low APC Cy7 + R7805 Ter119 very high PE CY7 +++ B7806 Death DAPI +++++ V4507 8


1 Cd3 high GFP ++ B5302 cd4 high APC CY7 ++++ R7803 cd8 high APC ++++ R6604 cd19 very low PE ++++ B5855 Ter119 very high Percp cy5.5 +++ B6956 Death DAPI +++++ V4507 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


Saturating

4

7

4

5/7

NonSaturating

01

FMO (fluorescence minus one) Contains all markers except one To discriminate positive vs negative populations


http://www.dartmouth.edu/~dartlab/?page=flow-cytometry

Name Rob SalomonPanel Name 2015 B/T Sep

Desired Outcome Separation of B cells from CD4 and CD8 Tcells

Instrument Canto II


1 cd3 high GFP ++ B5302 cd4 high APC CY7 ++++ R7803 cd8 high APC ++++ R6604 cd19 very low PE ++++ B5855 Ter119 very high Percp cy5.5 +++ B6956 Death DAPI +++++ V4507 8

PUTTING IT ALL TOGETHER:TEST YOUR COMPLETE PANEL

Panel Plus Controls and Analyse

Unstained controlAs negative controls – no antibody presentTo assess any autofluorescence

Compensation controlsSingle 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

PUTTING IT ALL TOGETHER:CONTROLS

Question : Which Populations is the Positive ?

Unstained controls Allow relative Determination of positivity


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.


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.bdbiosciences.com/research/mult icolor/spectrum_viewer/index.jsp

http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.html

http://www.bdbiosciences.com/research/multicolor/spectrum_viewer/index.jsp













?

spectral viewers

http://www.bdbiosciences.com/research/mult icolor/spectrum_viewer/index.jsp

http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.htmlUse the














?

Spectral overlap occurs when

fluorochromes excited by the

same lasers emit in similar

ranges.

B 530 B 5850%

20%

40%

60%

80%

100%

120%Effect of spectral overlap - Instrument View

Perc

enta

ge o

f Si

gnal

in

Det

ecto

r

COMPENSATION THEORY

Compensation is applied at the single event level

B 530 B 5850%

20%

40%

60%

80%

100%

120%

Signal from Com-pensation Controls

Axis

Titl

e

overlap

overlap

B 530 B 585020406080

100120

FITC bright

Sign

al S

tren

gth

B 530 B 5850

20406080

100120

FITC dull

Sign

al S

tren

gth

Compensation removes the signal spillover from one fluorochrome into any other parameter.

COMPENSATION THEORY

MFI pos population NTC1

= MFI neg population NTC1



………………………………..MFI pos population NTC

n = MFI neg population NTC

n

MFI = Media Fluorescent IntensityNTC = 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





………………………………..MFI pos population NTC

n = MFI neg population NTC

n

MFI = Media Fluorescent IntensityNTC = Non Target channelWhere

D= Fluorescence in detector

F= Fluorescence signalN = FL from detector #

N = FL in Detector #

EFFECT OF COMPENSATION

Digital compensation

doesn’t change the underlying

data it just allows us to interpret it

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

34

5a

5b

NumbersPercentages – parent and total MFI - Median/Mean Fluorescent intensityCV’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 scaleThere must be controls to show the relationship

between populations Instrument settings must be constantBe 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 interest3. Label Everything

4. Draw your plots5. Open you gate hierarchy6. Draw the Gates7. 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 parametersSSc & FScblue laser - 3x red laser - 1x

Event rate< 3, 000

CANTO I

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

8 parametersSSc & FScblue laser - 4xred laser - 2x

Event rate< 15, 000

CANTO II

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

10 parametersSSc & FScviolet - 2x blue laser - 4x red laser - 2x

Event rate< 10, 000

AMR FORTESSA

4 laser 405nm (violet)488nm (blue) 533nm (YG)633nm (red)

15 parametersSSC & FSCviolet - 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 parametersSSc & FSc& FSc PMTUV - 2xviolet - 6x blue - 2x YG -4xred - 3x

Event rate < 20, 000

ARIA IIU CELL SORTER

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

12 parametersSSc & FScviolet - 2x blue - 6x red - 2x


ARIA III CELL SORTER

4 laser 405nm (violet)488nm (blue) 561nm (YG)633nm (red)

18 parametersSSc & FScviolet - 6x blue - 2x YG - 5xred - 3x


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


2. Check and remove air bubbles

FLUIDICS SYSTEM

Wet cart

Sheath filter

Flow Cell Waste



3. Ensure flow cell is free from air and blockages

FLUIDICS SYSTEM

Wet cart

Sheath filter

Flow Cell Waste



3. Ensure flow cell is free from air and blockages

4. Ensure no air bubbles in line and waste height doesn’t change

INSTRUMENT POWER

INSTRUMENT STARTUP

SHEATH FILTER

FLUIDICS PRIME

1. Turn system on2. Remove air From Sheath Filter3. Perform software fluidics startup

Canto I, and Canto II

FLUIDICS PRIME

1. Turn system on2. Remove air From Sheath Filter3. Turn laser off (if possible)4. Prime 2 x ( no Tube)5. Run TDW for 1 min – or until no air

in waste lines6. Turn laser on.

Calibur LSRII / LSRII SORP

FLOW CELL

Low

Medium

High

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

LegendLaser intercept

Core Stream

FLOW CELL

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

Low

Medium

High

LegendLaser intercept

Core Stream

Laser focal plane Signal

spread

SAMPLE FLOW RATE CONTROL

OPTICS

Laser emission Laser delivery

Sample laser

interaction at flow cell

Emission collection

Spectral separation

Ensure lasers are on - software LSRII SORP ORHardware

OPTICS


Sample laser


Emission collection

Spectral separation


Ensure Clean Flow cell

OPTICS


Sample laser


Emission collection

Spectral separation


Ensure Clean Flow Cell

Achieved by filter selection

OPTICS: LASERS

OPTICS: FLOW CELL

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

col lecting emitted or

scattered l ight

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 ?

DichroicMirror

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 CY7B 670 PE CY5

PerCPB 610 Dichroic

only B 575 PEB 530 FITC/ GFP488/10 SSC

UNDERSTANDING PMT ARRAYS Position

Wave length

ABCDEF

A


Wave length

A >488nmBCDEF

A


Wave length

A >488nmBCDEF

A

B

UNDERSTANDING PMT ARRAYS position

Wave length

A >488nmB >735nmCDEF

A

B


Wave length

A >488nmB >735nmCDEF

A

B

C


Wave length

A >488nmB >735nmC 750-810nmDEF

A

B

C


Wave length

A >488nmB >735nmC 750-810nmDEF

A

B

C

D


Wave length

A >488nmB >735nmC 750-810nmD 488-735nmEF

A

B

C

D


Wave length

A >488nmB >735nmC 750-810nmD 488-735nmEF

A

B

C

D

E


Wave length

A >488nmB >735nmC 750-810nmD 488-735nmE 655-735nmF

A

B

C

D

E


Wave length

A >488nmB >735nmC 750-810nmD 488-735nmE 655-735nmF

A

B

C

D

E

F


Wave length

A >488nmB >735nmC 750-810nmD 488-735nmE 655-735nmF 670-735nm

A

B

C

D

E

F

CONFIGURATION DOCUMENTS

UNDERSTANDING THE PMT

Detector or PMT

Amplification Voltage

Electron Cascade Digitisati

on and processing

http://sales.hamamatsu.com/assets/applications/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 ???



Mid Voltage

Low voltage

flow cytometry training: introduction day 1 session 2

Education