660 Fa09 Flow Cytometry

8/2/2019 660 Fa09 Flow Cytometry

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Flow C t om et r an d Flow C t om et r an d So r t i ng So r t i ng

Sum m er RainesSum m er RainesOct o b e r 5 t h , 2 0 0 9Oct o b e r 5 t h , 2 0 0 9 smr a ines@w isc .edu smr a ines@w isc .edu

At t i eA t t ie La bLa b

B io ch e m is t r y 6 6 0B io ch e m is t r y 6 6 0

Theory of flow cytometry and sorting

Equipment

Light Scatter

Fluorescence

es gn ng a ow cy ome ry exper men

Video example of a flow cytometry experiment

-

-

Measurement of multiple characteristics of a single

cell/particle simultaneously

Measures: size, shape, granulation (light scattering)

metabolic ro erties fluorescence

Measure 105 to 106 cells/minute

FACS (Fluorescence-activated cell sorting) separates cellsn o reusa e su -popu a ons

Bas ic Flow Cy t om ete r

Ultrasonic nozzle vibrator

Optics System

Laser

Light scatter Fluorescence

+/ +/+

scatt

er

ulation)

Intensity

/

/+

Forward scatter

(size)

Side

(gra

Red intensity

Gree


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FACS

Ultrasonic nozzle vibrator

Laser

+ DeflectionplatesCell suspension

FACS Exam p le Sor t i ng -ce l l s fo r t r an sp an tat on

79.2%

. .

ulin

79.2% 8.7%8.7%Ins

Insulin

Nature Biotechnology - 24, 1392 -1401 (2006)

Synaptophysin Synaptophysin

Laser

Side Scatter

Proportional to granulation

More granulation = higher sidescatter

Side Detector

Forward

Detector

Forward Scatter

Proportional to particle size/shape

Larger particles = higher forward scatter

600

800

atter Granulocytes

400

SideSc

0

200

Lymphocytes

onocy es

Forward Scatter

Lymphocyte Monocyte Granulocyte


3/8

550 Long pass 635 Long pass 735 Long pass

LaserDichroic

mirrors585nm

700nm

530nm

Bandpass

Filters

525/15 580/20 700/20

Greenchannel

Orangechannel

Redchannel

Multiple-fluorochromes measured simultaneously

Determine labeling method

antibodies against cell surface vs. intracellular antigens

dyesIdentify fluorescence properties

Necessary lasers and filters

Fluorophore combinations to reduce spectral overlap

ControlsBiological controls (un-treated, non-transfected, etc)

Isotype controls - reduce background fluorescence

Compensation controls - reduce spectral overlap

Prepare cells

~1 x 10 cells/mL density 10,000 total cells counted per sample

3 technical replicates of 3 biological replicates

Em ission Spect r a l Over lap

PE

APC

PerCP

1000

nsity

PI800

600enceInt

FITC400

Fluores

200

Emission

500 550 600 650 700

Wavelength

Fluorophores must each have distinct emission

spectra to view in the same cell

Controls for non-specific binding of the labeled antibody

Test Antibodies

- -

Rabbit anti-human ProteinB-Green (IgG)

Y

Isotype Control Antibodies

#1 - Mouse non-specific IgG-Orange

#2- Rabbit non-specific IgG-Green

Note: If secondary antibodies are labeled, no-primary

antibody controls are the proper isotype controls


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Controls for spectral overlap of fluorophores

Test Antibodies

- -- -


Y

Compensa t ion

PE

APC

PerCP

1000

nsity

PI800

600enceIn

t

FITC400

Fluores

200

Emission

500 550 600 650 700Wavelength

Fluorophores must each have distinct emission

spectra to view in the same cell


Test Antibodies

- -


Y

Compensation Control Antibodies

#1 - Rabbit non-specific IgG-Green +

- -

#2- Mouse non-specific IgG-Orange +

Rabbit anti-human ProteinB-GreenYY

Com en sat ion Ex am le #1 - Rabbit non-specific IgG-Green +

Mouse anti-human ProteinA-Orange

YY

1000010000

1000

L2-585/PE

nge

1000

L2-585/PE

nge

10

FL2-H:F

Ora

10

FL2-H:F

Ora

1 10 100 1000 100

1

1 10 100 1000 100

FL1-H: FL1-530/FITC

1

Green

CompensatedUncompensated


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Com en sat ion Ex am le #2- Mouse non-specific IgG-Orange +

Rabbit anti-human ProteinB-Green

1000010000

100

1000

L2-585/PE

nge

100

1000

L2-585/PE

nge

10

FL2-H:F

Ora

10

FL2-H:F

Ora

1 10 100 1000 100

FL1-H: FL1-530/FITC

1

Green1 10 100 1000 100

FL1-H: FL1-530/FITC

1

Green

CompensatedUncompensated

Dat a Ana l ysi s / Presen ta t i on

Dot Plote

Orange

uoresce

nc

intensity

- Plots 2 parameters against oneanother

fl -

ForwardScatter

Histogram

Coun

t

- Plots 1 parameteragainst number of cells

Orange fluorescence intensity

-

ity

800

ter

1000

nce

Intens

400

SideScat

100

Fluoresce

0

200

1

10

Green

0 200 00 600 800 100

Forward Scatter0 200 400 600 800 100

Forward Scatter

e ec s nc popu a ons or ur er

data analysis or sorting

Type 2 Diabetes = insulin

need overcomes insulinproduction due to loss of-cells

Manipulate -cell growth =

www.webhealthindia.com/ ?tag=nerves-system


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-

Cholecystokinin (CCK)

Gut-derived peptide

CCK? Up-regulated in miceresistant to Type 2

DiabetesCausative or compensatory?

Involved in cell cycle control?

www.herb4cancer.wordpress.com/ 2007/11/

Human cadaver pancreas

Isolate islets

Infect with -gal (control) or CCK adenovirusWidth of cells

Dissociate islets into individual cells

Insulin stainin

Isolate -cells

Examine cell cycle progression between -gal and CCK-treated -cells

To stain for insulin (Identify -cells)Guinea i anti-human insulin I G

Goat anti-guinea pig IgG-FITC

Y

To stain for DNA content (Identify cell cycle stage)

Propidium Iodide (DNA dye)

(Go/G1)

(G2/M)

(S)

http://www.barrettsinfo.com/figures/fig6a1_2.gif


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Controls for non-s ecific bindin of the labeled antibod

Test Antibodies

Guinea pig anti-human Insulin IgG

Goat anti-guinea pig IgG-FITC

Y

Isotype Control (No-primary antibody)

Goat anti-guinea pig IgG-FITC ALONE

Note: No isotype control required for propidium iodide,

as it is antibody-independent

Compensa t ion

1000ity

PI800

ceIntens

FITC

600

400uorescen

200

issionFl

500 550 600 650 7000E

m

Wavelength


Test Antibodies

Guinea pig anti-human Insulin IgGGoat anti-guinea pig IgG-FITC

Propidium Iodide

Compensation Control Antibodies

a pig non-specific IgG Goat

ea pig IgG-FITC + Propidium

Iodide

#2 - Guinea pig anti-human Insulin IgG

-guinea pig IgG-FITC + NO

Propidium Iodide

Y

- - - .

vs. G2/M?

How do these percentages change upon over-expression of CCK?

Does CCK promote or inhibit-cell cycle progression?


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Cell sortin

Ion levels / flux

-

Enzyme activity

Cell proliferation or death

Cell c cle

UW CCC Flo w Faci l i t yRm K4 / 535 UW Cl in i ca l Sc iences Cen te r , McArd le Room 118

Kathy Schell: 263-0313; [email protected]

www.cancer.wisc.edu/uwccc/services flow.as_

FACScan

488nm ar on laser

FACS Vantage SE

2 coherent lasers ar on Innova-90

3 fluorescence channels

technician or user operated

, ,

krypton Innova-302, HeNe lasers


FACSCalibur

488nm argon and 633nm HeNe

technician operated

LSRII

lasers


, ,


technician operatedec n c an or user opera e

Flow Cytometry

Protein Expression?

Protein u - or down- re ulation?

Sub-cellular localization?

Protein binding partners?

Post-translational modification?

Protein/Metabolic profile?

Disease diagnostics?

Antibodies against foreign proteins?

Sum m ary o f Techn iques

Excellent Ver ood Good Fair Poor

Easy CheapHigh

through-put Quantitative

Flow Cytometry

Documents

660 Fa09 Flow Cytometry