What is Flow Cytometry?

Flow Cytometry uic

Introduction to Flow CytometryIGC Workshop

Multicolor Flow Cytometry

Rui GardnerIGC – April 03, 2013

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

HyperCyt

LSR Fortessa

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

Know Your Instrument (LSR Fortessa)

LSR Fortessa Optical Configuration

442 nm (BV)

561 nm (YG)488 nm (Blue)

Know Your Instrument (LSR Fortessa)

YELLOW GREEN (561 nm)

YG:561nm

PE-TexasRedPImCherrymRaspberrymplum

630/30 780/60

670/40 590/30

610LP 740LP

650LP

PE-Cy7

630/75 or 590LP in position AFor mCherry detection only

PE-Cy5, PE-A647mPlum

PERFP, DsReddTomatomOrange

Know Your Instrument (LSR Fortessa)

445/15

470/20

577LP

455LP

SSC(BV)

CFP442 nm (BV)

BLUE VIOLET (442 nm)

Know Your Instrument (LSR Fortessa)

BLUE (488 nm)

Blue:488nm

FITCAlexa 488GFPYFP

530/30 780/60

690/40 488/10

502LP 740LP

655LP

PE-Cy7

PE-Cy5

SSC

Know Your Instrument (LSR Fortessa)

Blue:488nm

GFP

510/20 780/60

540/30 488/10

502LP 740LP

525LP

PE-Cy7

YFP

SSC

BLUE (488 nm)

Measure GFP and YFP simultaneously

Choosing The Right Fluorochromes

Adapted from Holden and Trotter (Winter 2006) “Selecting Reagents for Multicolor Flow Cytometry” BD Hotlines newsletter, 11: 31.-34

Rule 1Choose the Brightest Fluorochromes

Fluorochromes (Stain Index)

Brightest Fluorochrome = Highest Stain Index

W2

W1

D

Stain Index (SI) =D/W

MFIPOSITIVE MFINEGATIVEL̶

2 × rSDNEGATIVE

Stain Index =

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

Rule 2Minimize Potential Spillover

Spillover (Minimize spillover)

A single fluorochrome can be detected in more than one channel

Spectral Overlap Correcting spillover

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/

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

Roederer, M. 2002. Compensation in Flow Cytometry. Current Protocols in Cytometry. 1.14.1–1.14.20

Spillover (Minimize spillover)

Slide taken from presentation: Mario Roederer, “Compensation: Basic Principles”, Monday, June 20, 2011

Rule 3Reserve brightest fluorochromes for

“dim” antibodies and vice-versa

Colors and Antibody Specificities(Reserve bright labels for dim antibodies)

Single Stain Controls

CD25-PE Fluorescence

CD25-PE Fluorescence

CD4-

Alex

a488

Flu

ores

cenc

eCD

4-Al

exa4

88 F

luor

esce

nce

CD4-

PE F

luor

esce

nce

CD4-

PE F

luor

esce

nce

CD25-Alexa488 Fluorescence

Single Stain Controls

CD25-Alexa488 Fluorescence

Rule 4Avoid spillover from bright populations into detectors requiring high sensitivity

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

Rule 5Take steps to avoid tandem dye degradation

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 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

Pacific Orange Pacific Orange

Pacific Blue Pacific Blue Pacific Blue Pacific Blue

Fluorochrome choices for 5 or more colors (Recommended by IGC)

Completely Outdated!

What is Flow Cytometry?

Flow Cytometry uic

Introduction to Flow CytometryIGC Workshop

Multicolor Flow Cytometry (end)

Rui GardnerIGC – Aprli 03, 2013