660 fa09 flow cytometry
TRANSCRIPT
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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
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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
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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
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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
- -- -
Rabbit anti-human ProteinB-Green (IgG)
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
Controls for spectral overlap of fluorophores
Test Antibodies
- -
Rabbit anti-human ProteinB-Green (IgG)
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
Controls for spectral overlap of fluorophores
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
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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
8 fluorescence channels
FACSCalibur
488nm argon and 633nm HeNe
technician operated
LSRII
lasers
4 fluorescence channels
, ,
11 fluorescence channels
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