Flow cytometry

Principles Cytometers Fluorochromes Applications

What is flow cytometry?

Flow cytometry is a method of measuring and quantify multiple physical and chemical characteristics of particles by optical means
Bone marrow cells Blood cells Bacteria Chromosomes Embriod bodies

Principles
Cells and particles must be in suspension Cells or other particles interact with a light beam as they pass by single file in a liquid stream. Interaction with the light is generally measured as: light scatter and fluorescence (according to staining of the cells) If a fluorochrome is specifically and stoichiometrically bound to a cellular component, the fluorescence intensity will ideally represent the amount of that particular component, i.e., cell-surface receptor, DNA, intracellular receptor, RNA or chromosomes This combination of scattered and fluorescence light is picked up by the detectors. By analyzing fluctuations in brightness at each detector it is possible to deduce various facts about the physical and chemical structure of each individual particle

 These characteristics are analyzed individually as the flowing cells pass through a focused laser. Many different parameters can be distinguished Size Granulosity Different surface and intracellular molecules depending on the potentialities of the cytometer and the fluorescent markers Flow cytometry allows computer assisted analysis of different characteristics for cells in suspension Cells can be sorted (separation and purification) individually according to combinations of these parameters.

Cytometers

Aligns particles in a stream Reads physical characteristics and fluorescence of those particles through light beam (laser) Digitalize and records measured parameters Analysis results with an appropriate software

FACScalibur

FACSCanto

FACSAria

PMT +Filters
Laser Ar Ion 488 nm

Air Pressure Pump Waste tank Laser optics FSC Photo diode Flow chamber Sample Tube Dichroic Mirror
(for SSC detection)

Unidades do citmetro
FLUIDOS: Suspenso de partculas e Fluxo laminar OPTICA: Fonte de iluminao, Captao da disperso da luz e da fluorescncia, Filtrao da disperso e da fluorescncia. ELECTRONICA: Converso do sinal em valores analgico digitais. Aquisio, Processamento, Anlise e Armazenamento por computador

Fluorescence Excitation Spectra

Wavelength nM
700 650 600 550 500 450 400 300

RED

ORANGE

YELLOW

GREEN

BLUE

VIOLET

ULTRAVIOLET

He Ne Lasers 633-635 nM

Yellow Dye Lasers 588nM

Violet Lasers 405 nM Argon laser 488 nM

UV Lasers He/ Cd Krypon 325 nM 355 nM

Eexc> Eem exc< em

Optical system

FSC and SSC

Side scatter Foward scatter

Argon Laser

FSC correlates with the cell volume

SSC depends on the inner complexity of the particle (i.e. shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness).

FSC and SSC detectors

Forward scatter tends to be more sensitive to surface properties of particles (e.g., cell ruffling) than side scatter. It can be used to distinguish live from dead cells

Laser
Sensor de FS

Sensor SS 900 Side scatter tends to be more sensitive to inclusions within cells than forward scatter can be used to distinguish granulated from non-granulated cells

FSC vs SSC

Complexidade

Granulocytes Monocytes Lymphocytes

0 256 512 768 1024

FSC-Height -> 1016142A.001

Dimenso

Fluorescence detectors
(FL1, FL2,)

Laser
Sensor de FS

Detectores de Fluorescncia

Dyes used for phenotypic analysis and excitation wavelength

Violet laser 405 nM Alexa Fluor 405 Alexa Fluor 430 Cascade Blue (Cascade Yellow)* Argon laser 488 nM FITC Alexa Fluor 488 R-PE PE-Texas Red (ECD) PE-Cy5 PerCP PerCP-Cy5.5 PE Cy7 Yellow dye laser 588 nM Rhodamine Texas Red Cascade yellow Cy5.5-APC (APC)* (APC-Cy7)* He Ne laser 647 nM APC Alexa Fluor 647 APC-Cy7

*Some dyes (ex. APC, APC-Cy7) can be activated over a broad range of different excitation wavelengths

Common Laser Lines

350
300 nm

457 488 514

400 nm 500 nm

610 632
600 nm 700 nm

PE-TR Conj. Texas Red PI Ethidium PE FITC cis-Parinaric acid

Available Fluorochromes (Visible Light Emission)

Fluorochrome
FITC Cy3 R-PE (PE) PE-Texas Red Pe-Cy5 PerCP PerCP/Cy5.5 PE-Cy7 Texas Red APC Cy5 APC-Cy7

Laser (nm)
488 488 488 488 488 488 488 488 595 633, 635 633, 635 633, 635

Emission (nm)
525 565 575 615 667 675 695 767 615 660 667 767

Tandem fluorochromes: PE-Cy7

The Visible Spectrum Infrared Light
700 600 500 Wavelength (nm) 400

Ultraviolet Light

488 nm Argon Light

Cy7 Cy7

767 nm, FL5

575 nm, FL2 Cy7 R-Phycoerythrin, Chains: A, B, K, and L

488 nm PE 575 nm Cy7 767 nm

767 nm, FL5

Emitted Fluorescence Intensiy Binding Sites

FITC

FITC FITC

FITC FITC

FITC Numero de eventos

FITC FITC FITC FITC

Intensidade de Fluorescncia

Acquisition and data analysis

 Clearly identify the question/s you are asking:

Is the percentage of cells expressing antigen of interest the most important point? or the relative level of expression? or the absolute number of cells with a particular characteristic?

Data measured in flow cytometry are visualized in graphs. The most commonly used are:

One parameter: - histogram Two parameters: - dot-blot diagrams and their derivatives - contour plot - density plot - pseudocolor plot - zebra plot

Single parameters can be displayed as a histogram.

Histograms display relative fluorescence or scattered light signals plotted against the number of events

Two-parameter (or dual-parameter) data can be displayed in two dimensions using dot, density or contour plots.
Dot SSC Scatter Density Pseudo Color Contour Plot

FSC
Relative levels of two parameters which were collected at the same time

Negative control Test Sample

Flow cytometry data analysis

One-colour histogram plot of CD8 expression by peripheral blood lymphocytes. Approximately 38% of events fall between the marker boundaries, and are therefore regarded as CD8+

Population of CD34 + 'stem cells plotted against side scatter.

Dual-parameter dot-plot CD8 expression on PB lymphocytes. Relationship between CD8 and the T-cell marker CD3. The CD8 + population contains two CD3-defined sub-populations (CD3+ and CD3-).Tthe CD3- fraction, expresses CD8 at a lower intensity than the CD3+ fraction.

256

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1024

10 0

10 1

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10 0

10 1

10 2

10 3

10 4

FSC-Height -> 1016142A.002

CD3 PerCP -> 1016142A.002

CD4 FITC -> 1016142A.002

256

512

768

1024

10 0

10 1

10 2

10 3

10 4

10 0

10 1

10 2

10 3

10 4

10 0

10 1

10 2

10 3

10 4

FSC-Height -> 1016142A.003

CD19 FITC -> 1016142A.003

CD56 PE -> 1016142A.003

CD19 FITC -> 1016142A.003

Gating
ungated Gated on R3

SSC

FSC

CD8
CD4- timcitos, Th moncitos e macrfagos CD8- Tc e NK

CD4

Gating complex subpopulations

Lymphocyte gate
SSC Scatter CD 4 PE

CD 4 gate
CD 45 RA Cy5

Analysis

FSC Scatter

CD8 FITC

L Selectin PE-Cy5

The most common acquisition error is insufficient data points When acquiring samples with rare events of interest acquisition should be set in order to get significant numbers of them.

UNIDADE DE CITOMETRIA DE FLUXO

Laser Laser

Pulse Height

Pulse Area

Pulse Height

Pulse Area

Pulse Width

Pulse Width

Laser

Laser

Pulse Height

Pulse Area

Pulse Height

Pulse Area

Pulse Width

Pulse Width

UNIDADE DE CITOMETRIA DE FLUXO

Doublets
Pulse Area

G2/M S G1

Pulse Width G1
Pulse Height Pulse Height

G2/M
Pulse Height

Doublets

Pulse Area

Pulse Area

Pulse Area

Pulse Width

Pulse Width

Pulse Width

UNIDADE DE CITOMETRIA DE FLUXO

G0-G1

G1

Cell Number

G2-M

S Fluorescence Intensity
of cells stained for DNA content

G2

UNIDADE DE CITOMETRIA DE FLUXO

Removal of cell clumps Ungated

R1

Gated Doublets

UNIDADE DE CITOMETRIA DE FLUXO

Algumas regras prticas a observar antes da anlise em citometria:

1. Conhecer as clulas a analisar 2. Antecipar as suas modificaes fsicas 3. Identificar outras populaes celulares presentes na amostra 4. Definir o conjunto de anticorpos a utilizar 5. Efectuar as combinaes de maneira racional 6. Seleccionar os fluorocromos mais adequados 7. Calibrar o citmetro de forma adequada para a experincia concreta 8. Utilizar um controlo interno (quando possvel ou relevante)

Adaptado

1. Conhecer as clulas a analisar

256

512

768

1024

FSC-Height -> 1016142A.001

2. Antecipate physical modifications

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FSC-Height -> 1016142A.001

FSC-Height -> 1016142A.001

Adaptado

UNIDADE DE CITOMETRIA DE FLUXO

3. Identify other cellular populations present in the sample

10 0

10 1

10 2

10 3

10 4

CD34 PerCP -> 896341C.002

10 0

10 1

10 2

10 3

10 4

10 0

10 1

10 2

10 3

10 4

CD45 APC -> 896341C.002

CD11b FITC -> 896341C.002

CD45- nave e T virgens CD11b- integrins CD13- ganulocytes, monocytes and BM

Adaptado

UNIDADE DE CITOMETRIA DE FLUXO

4. Definir o conjunto de ACs a utilizar
TRANSFORMED SSC ->

Combinaes devem incluir

CD20 PE ->

C 19 TC-> D

C 10 FITC-> D

1. 2.

Ac para identificao de linhagem (ex CD3 vs CD20) Ac que definam estadios de diferenciao diferentes

TRANSFORMED SSC ->

CD20 PE ->

C 19 TC-> D

C 10 FITC-> D

CD10- pr-B CD20- todas as clulas da linhagem B (inactivo nos plasmcitos)

CD19 Tricolor

CD20 PE (B)
10 0

10

10 1

10 2

10

10 4

10 1

10 2

10 3

10 4

CD10 FITC -> (pre-B)

CD10 FITC -> (pre-B)

Adaptado

N de Ac = n de fluorescncias disponveis?
5. Efectuar as combinaes de maneira racional

Exemplo 5 Ac / 3 FL:
CD8+CD19 / CD3+CD56 / CD4

CD3 + CD56 PE -> CD8 + CD19 FITC ->

CD3- cels T CD4- Th, timcitos, moncitos e macrfagos CD8- Tc e NK CD19- componentes do co-receptor de B CD56- NK, subconj de T, adeso

Adaptado

N de Ac = n de fluorescncias disponveis?

Exemplo 7 Ac / 4 FL:
CD8 + L / CD56 + k / CD19 + CD4 / CD3

Relative Fluorescent Intensity of Selected Mouse Anti-Human CD4 Conjugates

6. Seleccionar os fluorocromos mais adequados

Ex. Avaliar a expresso de CD4 / CD8 / CD45 em linfocitos Fluorescncias disponveis: FITC / PE / PE-Cy7 / APC-Cy7

PE

CD45

PE

CD4

PE

CD8
Mesmo fluorocromo vs diferentes Ag
Adaptado

Relative Fluorescent Intensity of Selected Mouse Anti-Human CD4 Conjugates

CD4

Isotype Control

FITC

Positive Peak Mean: 40.90 Negative Peak Mean: 2.99 Signal / Noise: 13.68 Positive Peak Mean: 195.76 Negative Peak Mean: 2.60 Signal / Noise: 75.29 Intensity vs. FITC: 4.79 x Positive Peak Mean: 88.32 Negative Peak Mean: 3.29 Signal / Noise: 26.84 Intensity vs. FITC: 2.16 x Positive Peak Mean: 94.18 Negative Peak Mean: 3.34 Signal / Noise: 28.20 Intensity vs. FITC: 2.30 x

PE

PE-Cy7

APC-Cy7

Data acquisition courtesy of Dr. Eric D. Wieder and Lisa Gibson, Gladstone Institute of Virology and Immunology, San Francisco, CA Immunology,

Relative Fluorescent Intensity of Selected Mouse Anti-Human CD8 Conjugates Isotype Control

CD8

FITC

Positive Peak Mean: 182.65 Negative Peak Mean: 2.76 Signal / Noise: 66.18

PE

Positive Peak Mean: 2646.21 Negative Peak Mean: 2.63 Signal / Noise: 1006.16 Intensity vs. FITC: 14.49 x Positive Peak Mean: 646.46 Negative Peak Mean: 3.21 Signal / Noise: 201.39 Intensity vs. FITC: 3.54 x Positive Peak Mean: 188.59 Negative Peak Mean: 3.47 Signal / Noise: 54.35 Intensity vs. FITC: 1.03 x

PE-Cy7

APC-Cy7

1Data acquisition courtesy of Dr. Eric D. Wieder and Lisa Gibson, Gladstone Institute of Virology and Immunology, San Francisco, CA Immunology,

Relative Fluorescent Intensity of Selected Mouse Anti-Human CD45 Conjugates

CD45

Isotype Control

FITC

Positive Peak Mean: 392.19 Negative Peak Mean: 2.74 Signal / Noise: 143.14 Positive Peak Mean: 1766.85 Negative Peak Mean: 2.59 Signal / Noise: 682.18 Intensity vs. FITC: 4.51 x Positive Peak Mean: 345.41 Negative Peak Mean: 2.66 Signal / Noise: 129.85 Intensity vs. FITC: .88 x Positive Peak Mean: 132.03 Negative Peak Mean: 2.56 Signal / Noise: 51.57 Intensity vs. FITC: .34 x

PE

PE-Cy7

APC-Cy7

1Data acquisition courtesy of Dr. Eric D. Wieder and Lisa Gibson, Gladstone Institute of Virology and Immunology, San Francisco, CA Immunology,

Seleco adequada de fluorocromos

Avaliar a expresso de CD4 / CD8 / CD45 em linfocitos Fluorescncias disponveis: FITC / PE / PE-Cy7 / APC-Cy7

Concluso CD45 FITC / CD4 PE / CD8 PE-Cy7

Also, use the brightest to detect faint markers
Adaptado

7. Cytometer Calibration
I. Adjust PMT voltages using unstained cells II. Adjust compensation for each fluorochrome

I. Adjust PMT voltages using unstained cells

FL1

Adjust PMT Voltage

FL1

SSC

FL2

FL2

FSC
Lymphocyte Gate

FL3

FL3

All within the 1st log scale

II. Ajust COMPENSATION for each fluorochrome

Compensation is the process by which the spectral overlap between different fluorochromes -spillover- is mathematically eliminated

Emission Spectra
100% Normalized Intensity

FITC

RPE

APC

PerCP

0% 400 500 600 Wavelength (nm) 700 800

Ideally:

Observed:

FITC compensation
FITC 530/30 Relative Intensity PE 585/42 PerCP 670/LP

FL2- % FL1

500nm 550nm 600nm 650nm 700nm

Wavelength (nm)

PE compensation eliminates FITC contribution to PE signal

Perform Compensation
Q1 Q2

PE

PE

FITC

FITC

Uncompensated vs compensated

PE

Perform Compensation
FITC

PE

FITC

PE compensation

FITC 530/30

PE 585/42

PerCP 670/LP

Relative Intensity

FITC

FL1 - %FL2 PE
500nm 550nm 600nm 650nm 700nm 750nm 800nm

FITC

PE

Wavelength (nm)

PerCP

FL3 - %FL2 PE PE

PerCP

The choice of antibody for compensation

FITC 530/30

PE 585/42

PerCP 670/LP

Relative Intensity

Compensation is Intensity Dependent!!

500nm 550nm 600nm 650nm 700nm 750nm 800nm

Wavelength (nm)

Uncompensated

Partially compensated

Fully compensated

FacsCalibur Compensation Matrix

Fluorescence -%Fluorescence =Spectral Overlap

FITC PE PE PerCP PerCP APC

PE FITC PerCP PE APC PerCP

FL1-FL2 FL2-FL1 FL2-FL3 FL3-FL2 FL3-FL4 FL4-FL3

Tubes required for compensation

Unstained cells Singly-stained cells for compensation Mix

Tubes required for regular acquisition

Unstained cells Isotype controls Each fluorochrome Mixes

Adaptado

Potential Applications of Flow Cytometry

. Cell activation status . Cell surface antigens (phenotyping) . Cell cycle distribution . Cell division . Apoptosis . Differentiation state . Cytokine secretion Cell sorting . Activation of signalling pathways Levels of intracellular reactive oxygen species Analysis of mitochondrial membrane potential Gene reporter (GFP) Intracellular calcium flux Flow Karyotype (single chromosome analysis)

Areas of utilization
Hematology Immunology Cancer biology Pharmacology Analyis of somatic-cell genetics Karyotype analysis Mechanisms of multidrug analysis

The application of fluorescence molecules, especially fluorescence-conjugated antibodies, the spectrum of applications has expanded

Cell activation status

FSC x SSC
Medium IL-7

23%

67%

Activation

IL7 induz a diferenciao de diferentes subpopulas tmicas IL7 funciona como um anti-apopttico
Joo Barata, adapt.

Activation
Cell surface antigens (phenotyping)

CD69, CD71, etc

CD69- B e T activadas, timcitos, neutrofilos, eosinfilos, plaquetas, NK CD71- todas as cels em proliferao. Marcador de activao

Joo Barata, adapt.

Differentiation state
Analysis of T cell differentiation state from nave to memory

Joo Barata, adapt.

Analysis of T cell development

Joo Barata, adapt.

CD4 PerCP

CD3 FITC

CD8 PE

CD4 PerCP

1- CD3- CD4- CD82- CD3- CD4+ CD83- CD3-/lo CD4+ CD8+ 4- CD3+ CD4+ CD8+ 5- CD3+ CD4+ CD86- CD3+ CD4- CD8+

TN ISP DP TP CD4 SP CD8 SP

CD8 PE
Joo Barata, adapt.

Cell Cycle Analysis

Cell cycle distribution

DNA content analysis - Propidium Iodide

In an ideal world.

# of events

# of Events

Increase in fluorescence intensity

Increase in Fluorescence Intensity

Joo Barata, adapt.

Cell Cycle Analysis

Cell cycle distribution

DNA content analysis - Propidium Iodide

# of events

# of Events

G1 G2/M

Increase in fluorescence intensity

Increase in Fluorescence Intensity

Joo Barata, adapt.

Cell Cycle Analysis

Bromodeoxyuridine (BrdU) method

DNA content analysis - Propidium Iodide plus BrdU staining

- Thymidine analog - Taken up by cycling cells - Usually in combination with Propidium Iodide - Allows to detect cycling S phase cells

Cell Cycle Analysis

Propidium Iodide plus BrdU staining

Anti-BrdU FITC

S Phase
# of E vents

G1

G2/M

200

400 FL3-H

600

800

1000

Increase in Fluorescence Intensity

Propidium Iodide
Joo Barata, adapt.

Cell Division
Tracking cell divisions with CFSE
CFSE: Carboxy-fluorescein diacetate, succinimidyl ester (fluorescein molecule containing a succinimidyl ester functional group and two acetate moieties) Diffuses freely into cells Intracellular esterases cleave the acetate groups converting CFSE into a fluorescent, membrane impermeant dye. Not transferred to adjacent cells. Retained by the cell in the cytoplasm Does not adversely affect cellular function During each round of cell division, the relative intensity of the dye is decreased by half.
Joo Barata, adapt.

Cell Division Tracking cell divisions with CFSE

INCREASING DILUTION OF CFSE

STAIN WITH CFSE

Cell Divisions

Joo Barata, adapt.

Day 0 Unstimulated

PHA Stimulation
D4 C
CD8

te a

Gat e
Joo Barata, adapt.

Apoptosis
CELL DEATH FSC x SSC

T-ALL
Medium
10
4

Thymocytes
Medium
10 4 SSC-H: SSC-Height (Log Scale) 10 3 SSC-H: SSC-Height (Log Scale)

100nM Rapa
10
4

100nM Rapa
10 4 10 3

SSC-H: SSC-Height (Log Scale)

10

SSC-H: SSC-Height (Log Scale)

Viability

10

10

10

10 2

10 2

10 1 27.6 27.6%

10 1 15.9 15.9%

10 1

26.2% 26.2

10 1

25.6% 25.6

10 0 0 10
4

10 0 200 400 600 800 FSC-H: FSC-Height 1000 10

4

10
0 200 400 600 800 FSC-H: FSC-Height 1000

10 0 200 400 600 800 FSC-H: FSC-Height 1000

0 10 4

200

400 600 800 FSC-H: FSC-Height

1000

10 4

Activation

SSC-H: SSC-Height (Log Scale)

SSC-H: SSC-Height (Log Scale)

SSC-H: SSC-Height (Log Scale)

10

10

10 3

10

10

10 2

10 1

37.4% 37.4

10 1

17.1% 17.1

10 1

26.1% 26.1

SSC-H: SSC-Height (Log Scale)

10 3

10 2

10 1

28.8 28.8%

10 0 0 200 400 600 800 FSC-H: FSC-Height 1000

10 0 0 200 400 600 800 FSC-H: FSC-Height 1000

10

10 0 200 400 600 800 FSC-H: FSC-Height 1000

200

400 600 800 FSC-H: FSC-Height

1000

Joo Barata, adapt.

Apoptosis
Propidium Iodide (fixed cells)

DNA degradation

Joo Barata, adapt.

Apoptosis
Annexin V plus propidium Iodide (non-fixed cells)

Joo Barata, adapt.

Apoptosis
Annexin V plus propidium Iodide

Joo Barata, adapt.

Apoptosis
Annexin V plus propidium Iodide

Joo Barata, adapt.

Apoptosis
Bcl-2 family members

(intracellular cytokine staining)

Fix and permeabilize

Add Antibody

Analyse by Flow Cytometry

Joo Barata, adapt.

Apoptosis
Bcl-2 family members Bcl-2

Joo Barata, adapt.

Apoptosis
Activated forms of caspases

Untreated

Etoposide

Flow cytometric analysis of Jurkat cells, untreated (blue) or etoposide-treated (green), using Cleaved Caspase-3 (Asp175) Antibody (Alexa Fluor 488 Conjugate).
Joo Barata, adapt.

Activation of signalling pathways

Akt MAPKs Stats etc, etc

Joo Barata, adapt.

Activation of signalling pathways

CD4 and CD8 distinguishable in a lymph node cell pop stim with IL-2 PFA/saponin protocol failed to detect P-STAT5 CD4+ have a heterogeneous response CD8+ respond uniformly Such heterogeneity would not have been detected by Western blot
Joo Barata, adapt.

Combining surface markers with Phospho-staining

Joo Barata, adapt.

Combining surface markers with Phospho-staining

Joo Barata, adapt.

Cell sorting
is a special application of flow cytometry

light source

Optical and/or electronic sensors

Flow sorting allows cells with preselected characteristics to be diverted from the stream and collected for further analysis

Flow cytometry can be used to separate individual chromosomes

Chromosomes are stained with fluorescent dye The dye taken up is porportional to chromosome size

A detector determines a particular chromosomes identity from its unique fluorescence

And signals a charge ring to apply a charge to the designated drops, which are deflected into a separate receptacle

Pierce 19.10

Flow karyotype of human chromosomes

Chromomycin A3 show preferential binding to GC-rich sequences Hoechst 33258 bind preferentially to AT-rich regions
Very high resolution, measured under low-speed sorting (15 to 35 chromosomes/sec). All chromosomes are resolved except numbers 9-12.

Future

More colors of fluorescence are being detected by cytometers Faster analysis and sorting rates More reagents More aplications Integration of molecular biology techniques

Available analysis software

CELLQuest WinList Flowjo WinMDI

http://flowcyt.cyto.purdue.edu/flowcyt/

Sites
www.flowcyt.salk.edu (flow cytometry on the web) http://jcsmr.anu.edu.au/facslab/analysis.html www.cytometry.org (discussion forums) http://www.bostoncytometry.org/pdf/Trotter2003.pdf (compensation)