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What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry Rui Gardner 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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Page 1: What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 28, 2010 Adapted from Holden

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

Page 2: What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 28, 2010 Adapted from Holden

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

Page 3: What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 28, 2010 Adapted from Holden

Know Your Instrument

Page 4: What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 28, 2010 Adapted from Holden

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

Page 5: What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 28, 2010 Adapted from Holden

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

Page 6: What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 28, 2010 Adapted from Holden

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

Page 7: What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 28, 2010 Adapted from Holden

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

)

Page 8: What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 28, 2010 Adapted from Holden

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

Page 9: What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 28, 2010 Adapted from Holden

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

Page 10: What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 28, 2010 Adapted from Holden

Choosing The Right Fluorochromes

Page 11: What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 28, 2010 Adapted from Holden

Fluorochromes (Stain Index)

Brightest Fluorochrome = Highest Stain Index

W2

W1

D

Stain Index (SI) =D/W

Page 12: What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 28, 2010 Adapted from Holden

Fluorochromes (Stain Index)

Freshly isolated lymphocytes, stained with anti-humanCD3 antibodies conjugated with various fluorochromes

Page 13: What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 28, 2010 Adapted from Holden

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

Page 14: What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 28, 2010 Adapted from Holden

Spillover (Minimize spillover)

A single fluorochrome can be detected in more than one channel

Spectral Overlap Correcting overlap

Page 15: What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 28, 2010 Adapted from Holden

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

Page 16: What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 28, 2010 Adapted from Holden

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

Page 17: What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 28, 2010 Adapted from Holden

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

Page 18: What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 28, 2010 Adapted from Holden

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

Page 19: What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 28, 2010 Adapted from Holden

Rules for choosing the right fluorochromes

Page 20: What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 28, 2010 Adapted from Holden

“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.

Page 21: What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 28, 2010 Adapted from Holden

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)

Page 22: What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 28, 2010 Adapted from Holden

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)

Page 23: What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 28, 2010 Adapted from Holden

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