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Colour Compensation in Flow Cytometry
January 31, 2013
Ian Dimmick (Newcastle University)
Graham Pockley (Nottingham Trent University)
Overview
Why do multicolour experiments?
Which Fluorochromes do I use?
What is spectral overlap and why is compensation needed?
2
compensation needed?
Procedure for compensation
Spill over examples
Spill over calculation procedure
Controls
Which Fluorochromes do I use?
What is spectral overlap and why is compensation needed?
Why do multicolour experiments?
3
compensation needed?
Procedure for compensation
Spill over examples
Spill over calculation procedure
Controls
3Ab+3Ab=18 Phenotypes
2
1 4
5
4
1
3
4
6
2
3
5
6
18 Phenotypes in 2 tubes for 6 antisera
6Ab’s=36 Phenotypes
1
2
1
3
1
4
1
5
1
6
5
36 Phenotypes in 1 tube for 6 antisera
2
3
2
4
2
5
2
6 4
3
4
5
3
5
3
6
4
6
5
6
Why do multicolour experiments?
What is spectral overlap and why is compensation needed?
Which Fluorochomes do I use?
6
compensation needed?
Procedure for compensation
Spill over examples
Spill over calculation procedure
Controls
When fluorochromes leave their excited state, they release energy in the form of a photon whose wavelength is longer that the excitation wavelength
7
Fluorescence Spectral viewers
8
Types of Fluorochromes(Non Tandem)
How bright is my Fluorochrome ?
FluorochromeAbs Emit
εεεεLaser FluorescenceQf
9
Specific wavelength(abs. Max)
Measured over entirespectra Values from 0.5-<1
Fluorescence intensity εεεε x QfTheoretical
Stain indices (SI=D/W)
Fluorochromea Stain index1PE-Cy5® 353
PE 3022APC 2782Alexa Fluor® 647 214
PE-Cy7® 1391PerCP-Cy5.5® 1073BD Horizon™ V450 85
10
“reserve the brightest fluorochromes for antigens with the lowest cellular concentration and vice versa”
3BD Horizon™ V450 853Pacific Blue™ 804Alexa Fluor® 488 73
Alexa Fluor® 700 614FITC 565APC-Cy7® 371PerCP 37
AmCyan 255APC-H7 24
Holden Maecker & Joe Trotter, Nature Methods 5, (2008)
How bright is the fluorochrome?
“Bright” = Good resolution sensitivity
Signal
Stain index
11
Signal
Cell
Types of Fluorochromes
Non Tandem
12
Tandem
Q dots
Non Tandem
13
Non Tandem
mean positive 57,382 mean positive 5,936
14
Compensation 525-450 14% Compensation 525-450 30.4%
15
Tandem dye breakdown
20mins RT 24 Hrs 4C 24 Hrs RT
16
20.98 22.75 34.59
Q Dots
17
Why do multicolour experiments?
What is spectral overlap and why is compensation needed?What is spectral overlap and why is compensation needed?
Which Fluorochromes do I use?
18
compensation needed?
Procedure for compensation
Spill over examples
Spill over calculation procedure
Controls
is compensation needed?
What is spectral overlap?
520/20 585/42 660/20
What does spectral
overlap look like on
my plots?
compensation objectives
PE
PE FITCPE
FITC PE
19
V4 b
450nm 500nm 550nm 600nm 650nm 700nm 750nm 800nm
FITC
FITC → FITC →
PE
→
PE →FITC
FITC
PE
500nm 550nm 600nm 650nm
FITC PE
Slide courtesy of Dr Rebecca Stewart
Multicolour panels, addressing dye vector, and laser/filter configuration
20
The more PMTs the closer the filters, the higher the compensation values
Compensation is Fi independentIn
tensity o
f sig
nal
G Y R FR
Compensation set – on spectral overlap values
Despite the decrease in fluorescence
intensity, the spillover still represents
10% of the total fluorescence
21
Inte
nsity o
f sig
nal
Wavelength
G-Y Y-G Y-R R-Y FR-Y FR-RSpillover on yellow detector
represents 10% of toral fluorescenceAn increase in the green detector
sensitivity amplifies the amount of
signal collected but the spillover for
yellow remains constant;
compensation decreases
Increased
gain on
green
detector
Compensation
Dependant on PMT Voltages
22
Available Lasers
Fluorochromes
Instrument filter configuration
Why do multicolour experiments?
What is spectral overlap and why is compensation needed?
Which Fluorochomes do I use?
23
compensation needed?
Spill over examples
Spill over calculation procedure
Controls
Procedure for compensation
FIT
vs
FITC
vs
PE
vs
FSC
vs
Procedure for compensation
No. fluorochromes No. plots
2 1
Example: Three-colour experiment CD3-FITC , CD8-PE and CD19-PerCPCy5.5®
Step 1: Construct your acquisition panel
vs
PE
vs
PerCPCy5.5®
vs
PerCPCy5.5®
vs
SSC
FITC PE PerCPCy5.5®
24
HAVE ALL FLUOROCHROME COMBINATIONS IN YOUR PANEL!HAVE ALL FLUOROCHROME COMBINATIONS IN YOUR PANEL!
2 1
3 3
4 6
5 10
6 15
7 21
8 28
9 36
10 45
AK7
Slide 24
AK7 We don't need this slide, it's the same as the one afterAris Krikelis, 13/12/2012
Procedure for compensation
Example: Three-colour experiment CD3-FITC CD8-PE CD19-PerCPCy5.5®
Step 3: Check for spectral overlap by running each fluorochrome separately and apply compensation if necessary
Tube 1: FITC only
Tube 2: PE only (remember to transfer your compensation values from your FITC tube!)
Look for the plots with PE on the axis: is there spectral overlap?
PE is spilling into FITC:
Adjust FITC -%PE
PE is spilling into PerCPCy5.5® :
Adjust PerCPCy5.5® -%PE
25
Look for the plots with PE on the axis: is there spectral overlap?
PE is spilling into FITC and PerCPCy5.5®
Procedure for compensation
Example: Three-colour experiment CD3-FITC CD8-PE CD19-PerCPCy5.5®
Step 4: Run your stained cell sample
26
Why do multicolour experiments?
What is spectral overlap and why is compensation needed?
Which Fluorochomes do I use?
27
compensation needed?
Procedure for compensation
Spill over calculation procedure
Controls
Spill over examples
Multicolour compensation Spill over
Where are my photons Qdot 605
28
Multicolour compensation Spill over
Where are my photons PE
29
Multicolour compensation Spill over
Where are my photons Pacific Blue™
30
Multicolour compensation Spill over
Where are my photons PeCy5®
31
Why do multicolour experiments?
What is spectral overlap and why is compensation needed?
Which Fluorochomes do I use?
32
compensation needed?
Procedure for compensation
Spill over examples
Controls
Spill over calculation procedure
Spectral overlap –calculation of Spill over
33
Compensation can be performed on cells if you have a bright signal, preferably as bright or brighter than anything you will encounter within your experiment
Spectral overlap –calculation of Spill over
34
Compensation can be performed on dual cell populations if you have a bright signal for each antigen, preferably as bright or brighter than anything you will encounter within your experiment and the antibodies are against mutually exclusive antigens
Spectral overlap –calculation of Spill over
Compensation beads
Polystyrene microparticles bind any kappa light chain-bearing immunoglobulin (mouse, rat, hamster and comp beads plus)
Kappa LCRantibody
35
Anti-Kappa
Each set of beads also contains negative beads with no binding capacity
Spectral overlap calculation of Spill over
AB. Fluorochrome combinations
The
Weak antigen : try to use a bright fluorochrome
Differentiate between compensation and data manipulation
If a fluorochrome is excited by more than one laser you
36
The Rules
are
If a fluorochrome is excited by more than one laser you need to evaluate if it is usable in a multicolour experiment
Always evaluate percentages of antigens by single colour controls, then check that the same percentage is achieved in your multicolor experiment
The lower the compensation values within your experiment, the more stable your experiment will be
Spectral overlap calculation of Spill over AB.
Fluorochrome combinations
Differentiate between compensation and data manipulation
37
Multiple laser excitation of fluorochromes
38
31%
9 colour well expressed antigen
39
10 colour S
ide s
catt
er
CD
4 P
e c
y7
CC
R7 P
e
CD
28 p
p 5
.5
CD
28 p
p 5
.5
CD3 Q605 CD8 APC-7 CD8 APC-7 CD27 APC CD27 APC
40
KLR
g1-A
488
PD
1-A
532
CD
57 A
350
CD
57 A
350
CD
57 A
350
CD
57 A
350
CD
4 P
e c
y7
CD
27 A
PC
A488A532Q605PercP5.5PeCy7APCAPC-Cy7Pac BlueA350A700
A488A532Q605PercP5.5PeCy7APCAPC-Cy7Pac BlueA350PE
CD27 APC CD27 APC KLRg1-A488 KLRg1-A488 CD45ra PB
CD27 APC KLRg1-A488 KLRg1-A488
CC
R7 P
eC
7
CD
4 A
700
Images provided by Jedrej Hoffmann, Ian Dimmick, Ioakim Spyridopoulos
Spectral overlap calculation of Spill over
AB. Fluorochrome combinations
Always evaluate percentages of antigens by single color controls, then
check that the same percentage is achieved in your multicolor experiment (Single [Single] Multicolour [M] ) diff%
S/M diff, %
A488 3.1
41
A532 0.3
Q605 3
PerCyp5.5 1.1
PeCy7 2.2
APC 3
A700 2.7
APC-CY7 2
Pac Blue 4
A350 1
Spectral overlap Calculation of Spill over
AB. Fluorochrome Combinations
High compensation
Values, increased
Compensation Stability
42
Instability,
Instrument Fi
monitoring critical low compensation
Values, decreased
Instability,
instrument Fi
monitoring less critical
Why do multicolour experiments?
What is spectral overlap and why is compensation needed?
Which Fluorochomes do I use?
43
compensation needed?
Procedure for compensation
Spill over examples
Spill over calculation procedure
Controls
Controls
Gold standard
The negative cells that are clearly distinguishable from the positive population in a stained sample
Negative cells
Instrument noise, autofluorescence
Isotypic Same protein concentration / Ig isotype /fluorochrome as Ab
44
controlSame protein concentration / Ig isotype /fluorochrome as Ab
Isotypic control (Bad)
Different protein concentration/Ig isotype /fluorochrome manufacturer
Isoclonic control
The use of unconjugated test specific antibody to block specific antigen sites to leave only non specific binding sites available to the conjugated test specific antibody
Isotype controls
When to use and when not to use
Picking the correct Isotype control
45
Picking the correct Isotype control
F:P ratio
Protein concentration considerations
Test Antibody+++++++++++NS+
46
No Antibody
Isotype Ctl Ig subtype matched Protein conc. MatchedInevitably will have Different tert. ConfigThan test Ab
-
NS+
Fluorescence minus one FMO on weak antigens
47
x
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No compensation needed with No compensation needed with Compensation required
54
No compensation needed with common FITC (or GFP) & PE combinations
No compensation needed with common FITC (or GFP) & PE combinations
Compensation required
Compatible with FITC/PE stained samples
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Blue laser co-excitation with FITC/GFP
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60
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