Multicolor Flow Cytometry

Report
What is Flow Cytometry?
Introduction to Flow Cytometry
IGC Workshop
Flow Cytometry
uic
Multicolor Flow Cytometry
IGC – 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
Cell Sorters
Analyzers
• Lasers
• Detectors and respective filters
FACScan
FACSCalibur
FACSAria
CyAn ADP
MoFlo
Know Your Instrument (FACScan)
FACScan Optical Configuration
Typical Fluorochromes
488
585/42
FL1
FL2
650LP
GFP
FITC
Alexa488
CFSE
FL3
488 nm
530/30
400
450
500
550
600
650
700
750
800
PE
PI
Cy3
PI
PE-Cy5
PE-Cy7
PerCP
PerCP-Cy5.5
7AAD
PE-Alexa610
Know Your Instrument (FACSCalibur)
FACSCalibur Optical Configuration
Typical Fluorochromes
488
585/42
670LP
FL1
FL2
FL3
GFP
FITC
Alexa488
CFSE
488 nm
530/30
400
450
500
550
600
650
700
750
PE
PI
Cy3
PI
PE-Cy5
PE-Cy7
PerCP
PerCP-Cy5.5
7AAD
PE-Alexa610
800
633
661/16
FL4
633 nm
APC
Cy5
Alexa647
400
450
500
550
600
650
700
750
800
Know Your Instrument (CyAn ADP)
CyAn ADP Optical Configuration
Typical Fluorochromes
405
530/40
400
450
500
488 nm
488
550
450
500
600
650
530/40 575/25 613/20
FL1
400
DAPI
Alexa 405
Pacific Blue
Violet 2
(FL7)
Violet 1
(FL6)
405 nm
450/50
550
FL2
700
680/30
FL3
600
750
700
FL5
750
642
500
550
600
650
PE
PI
PI
PE-Texas Red
PE-Alexa 610
PI
PE-Cy5
PerCP
PerCP-Cy5.5
800
APC-Cy7
(FL9)
642 nm
450
GFP
FITC
Alexa488
CFSE
750LP
APC (FL8)
665/20
400
800
750LP
FL4
650
Alexa 430
AmCyan
Pacific Orange
700
750
800
APC
Cy5
Alexa647
APC-Cy7
APC-H7
Alexa 700*
PE-Cy7
Know Your Instrument (FACSAria)
FACSAria Optical Configuration
Typical Fluorochromes
407
530/30
530/30
400
450
500
600
650
585/42 616/23
PE
FITC
488 nm
488
550
550
700
600
750
695/40
650
700
780/60
750
633
450
500
550
600
650
GFP
FITC
Alexa488
CFSE
PE
PI
PI
PE-Texas Red
PE-Alexa 610
PI
PE-Cy5
PerCP
PerCP-Cy5.5
800
APC-Cy7
780/60
APC
633 nm
660/20
400
Alexa 430
AmCyan
Pacific Orange
800
PE-Cy7
500
PerCP-Cy5.5
450
PE-Texas Red
400
DAPI
Alexa 405
Pacific Blue
Alexa 430
DAPI
407 nm
450/40
700
750
800
APC
Cy5
Alexa647
APC-Cy7
APC-H7
Alexa 700*
PE-Cy7
Know Your Instrument (MoFlo)
MoFlo Optical Configuration
#5
PE-Cy75
#6
795/50
PE-TxRed
APC
616/26
#4
SSC
Red
#8
D405/30
670/30
616/26
#1
FITC
H-Blue
530/40
#2
PE
488/10
#3
#7
585/40
670/40
Blue
Yellow
UV
mCherry
H-Red
#9
Choosing The Right Fluorochromes
Fluorochromes (Stain Index)
Brightest Fluorochrome = Highest Stain Index
D
W1
W2
Stain Index (SI) =D/W
Fluorochromes (Stain Index)
Freshly isolated lymphocytes, stained with anti-human
CD3 antibodies conjugated with various fluorochromes
Fluorochromes (Choose the brightest)
Stain Index of various anti-CD4 fluorochrome conjugates
measured on a BD LSR II
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
Spillover (Minimize spillover)
A single fluorochrome can be detected in more than one channel
Spectral Overlap
Correcting overlap
Compensation
Compensation is a mathematical subtraction to correct spectral overlap
A488true = A488measured - % 1PE true
0%%A488
5
10
15
20
% true
PE true = PE measured - 30
1000
Alexa488
100
10
1
http://www.drmr.com/compensation/index.html
http://http://igc-wiki.igc.gulbenkian.pt/
1
10
100
PE
1000
Colors and Antibody Specificities
(Reserve bright labels for dim antibodies)
Single Stain Controls
CD4-PE Fluorescence
CD4-Alexa488 Fluorescence
Single Stain Controls
CD25-Alexa488 Fluorescence
CD4-PE Fluorescence
CD4-Alexa488 Fluorescence
CD25-PE Fluorescence
CD25-PE Fluorescence
CD25-Alexa488 Fluorescence
Colors and Antibody Specificities
(Avoid spillover of bright cells into detectors of dim signals)
Sample
CD4-Alexa488
Fluorescence
CD4-Cy5 Fluorescence
CD4-Alexa488
CD4-Cy5 Fluorescence
Fluorescence
Single Stain Controls
CD4-Alexa488 Fluorescence
CD25-PE Fluorescence
CD25-PE Fluorescence
CD25-PE Fluorescence
Tandem Dyes
Watch out for degradation
TIME
APC Fluorescence
APC-50% PE-Cy5
PE-Cy5 Fluorescence
APC- 40% PE-Cy5
PE-Cy5 Fluorescence
PE-Cy5 Fluorescence
APC- 30% PE-Cy5
APC Fluorescence
PE-Cy5
PE-Cy7
APC-Cy7
APC Fluorescence
APC-H7
Rules for choosing the right fluorochromes
“Rules” for selecting Multicolor Panel
taken 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
Fluorochrome choices for 5 or more colors (Recommended by BD)
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
Recommended Multicor Panel
Fluorochrome choices for 5 or more colors (Recommended by IGC)
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
APC , Alexa 647 or Cy5
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
Alexa 700
Pacific Blue
Pacific Blue
APC-Cy7
APC-Cy7
AmCyan
AmCyan
Pacific Blue
Pacific Blue
What is Flow Cytometry?
Introduction to Flow Cytometry
IGC Workshop
Flow Cytometry
uic
Multicolor Flow Cytometry (end)
IGC – April 28, 2010
Adapted from
Holden and Trotter (Winter 2006) “Selecting Reagents for Multicolor Flow Cytometry” BD Hotlines newsletter, 11: 31.-34

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