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What is Flow Cytometry?
Flow Cytometry uic
Introduction to Flow CytometryIGC Workshop
Multicolor Flow Cytometry
Rui GardnerIGC – April 28, 2010
Adapted from Holden and Trotter (Winter 2006) “Selecting Reagents for Multicolor Flow Cytometry” BD Hotlines newsletter, 11: 31.-34
Outline
• Know Your Instrument• Optical Layout (lasers and filters)
• Choosing the right fluorochromes• Staining Index• Spillover• Compensation• Color Specificities and Tandem Dyes
• Rules for Multicolor Analysis
Know Your Instrument
Know Your Instrument
Reagent Selection starts with Instrument Configuration • Lasers• Detectors and respective filters
Anal
yzer
sCe
ll So
rter
s
FACSCaliburFACScan
MoFloFACSAria
CyAn ADP
Know Your Instrument (FACScan)
FACScan Optical Configuration
400 450 500 550 600 650 700 750 800
488530/30 585/42 650LP
FL1 FL2 FL3 GFPFITCAlexa488CFSE
PEPICy3
PIPE-Cy5PE-Cy7PerCPPerCP-Cy5.57AADPE-Alexa610
Typical Fluorochromes
488
nm
Know Your Instrument (FACSCalibur)
FACSCalibur Optical Configuration
Typical Fluorochromes
400 450 500 550 600 650 700 750 800
488530/30 585/42 670LP
FL1 FL2 FL3
488
nm63
3 nm
400 450 500 550 600 650 700 750 800
633661/16
FL4APCCy5Alexa647
GFPFITCAlexa488CFSE
PEPICy3
PIPE-Cy5PE-Cy7PerCPPerCP-Cy5.57AADPE-Alexa610
Know Your Instrument (CyAn ADP)
CyAn ADP Optical Configuration
GFPFITCAlexa488CFSE
PEPI
PIPE-Cy5PerCPPerCP-Cy5.5
Typical Fluorochromes
405
nm64
2 nm APC
Cy5Alexa647
400 450 500 550 600 650 700 750 800
488
nm
405450/50
400 450 500 550 600 650 700 750 800
400 450 500 550 600 650 700 750 800
488
642
Alexa 430AmCyanPacific Orange
DAPIAlexa 405Pacific Blue
PIPE-Texas RedPE-Alexa 610
PE-Cy7
APC-Cy7APC-H7Alexa 700*
530/40
530/40 575/25 613/20 680/30 750LP
750LP665/20
FL1 FL2 FL3 FL4 FL5
APC
(FL8
)
APC-
Cy7
(FL9
)
Viol
et 1
(FL6
)
Viol
et 2
(FL7
)
Know Your Instrument (FACSAria)
FACSAria Optical Configuration
GFPFITCAlexa488CFSE
PEPI
PIPE-Cy5PerCPPerCP-Cy5.5
Typical Fluorochromes
407
nm63
3 nm APC
Cy5Alexa647
400 450 500 550 600 650 700 750 800
488
nm
407450/40
DAPI
Alex
a 43
0
400 450 500 550 600 650 700 750 800
FITC PE
PE-T
exas
Red
PerC
P-Cy
5.5
PE-C
y7
400 450 500 550 600 650 700 750 800
APC
APC-
Cy7
488
633
Alexa 430AmCyanPacific Orange
DAPIAlexa 405Pacific Blue
PIPE-Texas RedPE-Alexa 610
PE-Cy7
APC-Cy7APC-H7Alexa 700*
530/30
530/30 585/42 616/23 695/40 780/60
780/60660/20
SSC
FITC
PE
PE-Cy75
#1
#2
#3
#4
#5
#6
#7
#8
#9
Blue
Red
488/
1053
0/40
585/
4061
6/26
95/5BS
555DLP
610DLP
645DLPPE-TxRed
795/50
670/40
APC
Know Your Instrument (MoFlo)
MoFlo Optical Configuration
H-Blue
H-Red
UV
670/30
565 D
CLP
D405/30
Yellow
mCherry
616/26
Choosing The Right Fluorochromes
Fluorochromes (Stain Index)
Brightest Fluorochrome = Highest Stain Index
W2
W1
D
Stain Index (SI) =D/W
Fluorochromes (Stain Index)
Freshly isolated lymphocytes, stained with anti-humanCD3 antibodies conjugated with various fluorochromes
Fluorochromes (Choose the brightest)
Reagent Clone Filter Stain Index
PE RPA-T4 585/40 356.3
Alexa 647 RPA-T4 660/20 313.1
APC RPA-T4 660/20 279.2
PE-Cy7 RPA-T4 780/60 278.5
PE-Cy5 RPA-T4 695/40 222.1
PerCP-Cy5.5 Leu-3a 695/40 92.7
PE-Alexa 610 RPA-T4 610/20 80.4
Alexa 488 RPA-T4 530/30 75.4
FITC RPA-T4 530/30 68.9
PerCP Leu-3a 695/40 64.4
APC-Cy7 RPA-T4 7801/60 42.2
Alexa 700 RPA-T4 720/45 39.9
Pacific Blue RPA-T4 440/40 22.5
AmCyan RPA-T4 525/50 20.2
Stain Index of various anti-CD4 fluorochrome conjugates measured on a BD LSR II
Spillover (Minimize spillover)
A single fluorochrome can be detected in more than one channel
Spectral Overlap Correcting overlap
A488true = A488measured - % PE true
PE true = PE measured - % A488true0 % 5 % 10 % 15 %20 %30 %
Compensation
Compensation is a mathematical subtraction to correct spectral overlap
http://www.drmr.com/compensation/index.htmlhttp://http://igc-wiki.igc.gulbenkian.pt/ 1 10 100 1000
1
10
100
1000
1 10 100 1000
1
10
100
1000
1 10 100 1000
1
10
100
1000
1 10 100 1000
1
10
100
1000
1 10 100 1000
1
10
100
1000
Alex
a488
PE
A488true = A488measured - % 1
Colors and Antibody Specificities(Reserve bright labels for dim antibodies)
Single Stain Controls
CD4-
PE F
luor
esce
nce
CD4-
PE F
luor
esce
nce
CD25-PE Fluorescence
CD25-PE Fluorescence
CD4-
Alex
a488
Flu
ores
cenc
eCD
4-Al
exa4
88 F
luor
esce
nce
CD25-Alexa488 Fluorescence
Single Stain Controls
CD25-Alexa488 Fluorescence
Sample
CD25-PE Fluorescence
CD4-
Alex
a488
Flu
ores
cenc
e
Single Stain Controls
CD4-
Alex
a488
Flu
ores
cenc
eCD
4-Al
exa4
88 F
luor
esce
nce
CD25-PE Fluorescence
CD25-PE Fluorescence
Colors and Antibody Specificities(Avoid spillover of bright cells into detectors of dim signals)
CD4-
Cy5
Fluo
resc
ence
CD4-
Cy5
Fluo
resc
ence
CD25-PE Fluorescence CD25-PE Fluorescence
Tandem Dyes
Watch out for degradation
APC Fluorescence APC Fluorescence APC Fluorescence
PE-C
y5 F
luor
esce
nce
PE-C
y5 F
luor
esce
nce
PE-C
y5 F
luor
esce
nce
TIME
PE-Cy5PE-Cy7 APC-Cy7 APC-H7
APC- 30% PE-Cy5 APC- 40% PE-Cy5 APC-50% PE-Cy5
Rules for choosing the right fluorochromes
“Rules” for selecting Multicolor Paneltaken from BD Biosciences
Rule 1: Choose the brightest set of fluorochromes for your particular instrument configuration.
Rule 2: Choose fluorochromes so as to minimize the potential for spillover.
Rule 3: Reserve the brightest fluorochromes for “dim” antibodies, and vice versa.
Rule 4: Avoid spillover from bright cell populations into detectors requiring high sensitivity for those populations.
Rule 5: Take steps to avoid tandem dye degradation, and consider its impact upon results.
Recommended Multicor Panel
5-color 6-color 8-color 10-color
FITC or Alexa 488 FITC or Alexa 488 FITC or Alexa 488 FITC or Alexa 488
PE PE PE PE
PE-Texas Red or PE-Alexa 610
PerCP-Cy5.5 PerCP-Cy5.5 PerCP-Cy5.5 PerCP-Cy5.5
PE-Cy7 PE-Cy7 PE-Cy7 PE-Cy7
APC , Alexa 647 or Cy5 APC , Alexa 647 or Cy5 APC , Alexa 647 or Cy5 APC , Alexa 647 or Cy5
Alexa 700
APC-Cy7 APC-Cy7 APC-Cy7
AmCyan AmCyan
Pacific Blue Pacific Blue
Fluorochrome choices for 5 or more colors (Recommended by BD)
Recommended Multicor Panel
5-color 6-color 8-color 10-color
FITC or Alexa 488 FITC or Alexa 488 FITC or Alexa 488 FITC or Alexa 488
PE PE PE PE
PE-Texas Red or PE-Alexa 610
PerCP-Cy5.5 or PerCP PerCP-Cy5.5 or PerCP PerCP-Cy5.5 or PerCP PerCP-Cy5.5 or PerCP
PE-Cy7 PE-Cy7 PE-Cy7
APC , Alexa 647 or Cy5 APC , Alexa 647 or Cy5 APC , Alexa 647 or Cy5 APC , Alexa 647 or Cy5
Alexa 700
APC-Cy7 APC-Cy7
AmCyan AmCyan
Pacific Blue Pacific Blue Pacific Blue Pacific Blue
Fluorochrome choices for 5 or more colors (Recommended by IGC)
What is Flow Cytometry?
Flow Cytometry uic
Introduction to Flow CytometryIGC Workshop
Multicolor Flow Cytometry (end)
Rui GardnerIGC – April 28, 2010
Adapted from Holden and Trotter (Winter 2006) “Selecting Reagents for Multicolor Flow Cytometry” BD Hotlines newsletter, 11: 31.-34
