Purdue University Cytometry Laboratories
Functional
assays Principles
and Methods
J Paul Robinson
These slides are on the PURDUE CYTOMETRY WEB SITE
http://flowcyt.cyto.purdue.edu
Presented at the
Polish Society for
Cytometry Meeting,
Gdansk, Poland,
October 18, 1998
Poland taken from
Comsat C1 satellite
October 15, 1998 you can clearly see
Warsaw in the
center. Top centerleft is Gdansk.
Gdansk
Warsaw
Krakow
Gdansk
The goals of this presentation are:
• To identify the nature of functional assays in
Cytometry
• To expand on how they operate
• To discuss the advantages and disadvantages of
each
• To discuss the application of these assays
Kinetics
Principle of Time Measurements
• Live cells can be measured as easily as dead cells
• You only need a small number of cells in a changing environment
• End point assays can describe the activity of the cell
Cellular Functions
• Cell Viability
• Phagocytosis
• Organelle Function
– mitochondria, ER
– endosomes, Golgi
• Oxidative Reactions
–
–
–
–
Superoxide
Hydrogen Peroxide
Nitric Oxide
Glutathione levels
• Ionic Flux Determinations
–Calcium
–Intracellular pH
• Membrane Potential
• Membrane Polarization
• Lipid Peroxidation
What do we measure?
TIME
Fluorescent Indicators
How the assays work:
• Superoxide: Utilizes hydroethidine the sodium borohydride reduced
derivative of EB
• Hydrogen Peroxide: DCFH-DA is freely permeable and enters the cell where
cellular esterases hydrolyze the acetate moieties making a polar structure
which remain in the cell. Oxidants (H2O2) oxidize the DCFH to fluorescent
DCF
• Glutathione: In human samples measured using 40 M monobromobimane
which combines with GSH by means of glutathione-S-transferase. This
reaction occurs within 10 minutes reaction time.
• Nitric Oxide: DCFH-DA can also be used as an indicator for nitric oxide in a
manner similar to H2O2
Organelle Function
•
•
•
•
Mitochondria
Endosomes
Golgi
Endoplasmic Reticulum
Rhodamine 123
Ceramides
BODIPY-Ceramide
DiOC6(3) Carbocyanine
DCFH-DA
DCFH
DCF
2’,7’-dichlorofluorescin diacetate
O
O
CH3-C-O
O
O-C-CH3
Cl
2’,7’-dichlorofluorescin
Cl
H
COOH
O
HO
Cellular Esterases
Cl
OH
Fluorescent
Cl
H
COOH
Hydrolysis
2’,7’-dichlorofluorescein
O
HO
O
H2O2
Cl
Cl
H
Oxidation
DCFH-DA
COOH
Neutrophils
DCFH-DA
80
Monocytes
60
H 2O 2
counts
DCFH
PMA-stimulated PMN
Control
40
20
DCF
Lymphocytes
0
.
1
1
100
log FITC 10
Fluorescence
1000
Hydroethidine
HE
EB
H2N
NH2
H
N
O2-
H2N
NH2
N + Br
CH2CH3
-
CH2CH3
Phagocytic Vacuole
NADPH Oxidase
NADPH
O2
HE
O2-
NADP
SOD
O 2H2O2
DCF
H2O2
OH-
Example: Neutrophil Oxidative Burst
DCF
Both these images are cells stained to measure for
H2O2 production.
Chondrocytes
Neutrophil
Some examples of rapidly changing
antigen expression systems
Endothelial
Adhesion Molecules
P-selectin (CD62P)
E-selectin (CD62E)
Neutrophil
Adhesion Molecules
L-selectin (CD62L)
Neutrophil Counter Ligand
s-Lex (CD15s)
s-Lex, CD66, L-selectin, b2 integrins
Endothelial Counter Ligand
s-Lex (CD15s)
CD11a/CD18
ICAM-1 (CD54), ICAM-2 (CD102)
CD11b/CD18
ICAM-1 (CD54), [iC3b, fibrinogen, factor X]
CD11c/CD18
?, [iC3b, fibrinogen]
a
b
c
d
TNFa
IL-1
Neutrophils
Endothelial Cells
L-selectin &CHO ligands
(e.g. sLex)
CD11b
E-selectin & P-selectin
BACTERIAL
INFECTION
ICAM-1
The circulating neutrophil (a) and the initiation of rolling (b) as molecular tethers are formed between
selectin and CHO ligands on neutrophils and endothelial cells. If an adequate number of tethers are
formed, the neutrophil completely decelerates and with chemotactic stimulation of the neutrophil, Lselectin is rapidly shed while other receptors like E-selectin, CD11b and ICAM-1 are up-regulated by
cytokines and other inflammatory mediators (c). Firm neutrophil/endothelial cell adhesion is mediated by
CD11b and ICAM-1 and is followed by emigration of the neutrophil through the endothelium (d).
? (NOO-)
H2O2
O2NO.
-
OH-
-
M
+ NO.
+
+
+
+
VCAM
+
NFkB
+ ROS
+
+
Bradykinin
TNFa
Known and unknown interactions between neutrophils and
endothelial cells. Nitric oxide (NO.) and reactive oxygen species
(ROS) are produced by both neutrophils and endothelial cells thus
the interaction between these reactive species becomes very
complicated.
ICAM-1
E-selectin
P-selectin
CD11b
Mmembrane damage
conjugated dienes
+ stimulatory effect
- inhibitory effect
Oxidative Reactions
•
•
•
•
Superoxide
Hydrogen Peroxide
Glutathione levels
Nitric Oxide
Hydroethidine
Dichlorofluorescein
Monobromobimane
Dichlorofluorescein
Rat Pulmonary Artery Endothelial Cells
Oxidization via H2O2
Periodicity of Fluorescence
Meridian UltimaTM Analysis
Purdue University Cytometry Laboratories
Macrovascular Endothelial Cells in
Culture
0
Time (minutes)
60
Confocal System
Culture System
top view
Step 1: Cell
Culture
Step 2: Cell
Wash
Step 3: Transfer to LabTek plates
side
view
Step 4: Addition of DCFHDA, Indo-1, or HE
1
2
3
4
5
6
7
8
170 M coverslip
stimulant/inhibitor
added 37o heated
stage
oil
immersion
objective
confocal microscope
Hydrogen peroxide measurements with DCFH-DA
1
2
3
4
Change in fluorescence was measured
using Bio-Rad software and the data
exported to a spread sheet for analysis.
5
525 nm
Step 6B: Export data from measured
regions to Microsoft Excel
% change (DCF fluorescence)
Step 7B: Export data from Excel data
base to Delta Graph
2000
1800
1600
1400
1200
1000
800
600
400
200
0
cell 1
cell 2
cell 3
cell 4
cell 5
0
500
1000 1500 2000 2500 3000
Time in seconds
Superoxide measured with hydroethidine
cell 1
Change in fluorescence was measured
using Bio-Rad software and the data
exported to a spread sheet for analysis.
cell 3
cell 4
cell 2
cell 5
Export data from Excel data
base to Delta Graph
%change (DCF fluorescence)
Export data from measured
regions to Microsoft Excel
1800
1600
1400
1200
1000
800
600
400
cell 1
cell 2
cell 3
cell 4
200
0
cell 5
-200
200 400
600 800 1000 1200 1400 1600 1800
Time in seconds
Promyelocyte
Metamyelocyte
Myelocyte
Neutrophil
Padma Narayanan figures hl60.ppt
e1
Change in Mean DCF Fluorescence
36
0 ng/ml PMA
30
8 ng/ml PMA
24
50 ng/ml PMA
18
d1
e
12
c1
b1
6
c
a
a
d
b
0
0 HOURS
24 HOURS
48 HOURS
72 HOURS
96 HOURS
Change in Mean DCF Fluorescence
36
0 HOURS PMA (8 ng/ml)
30
0 HOURS PMA (50 ng/ml)
96 HOURS PMA (8 ng/ml)
24
96 HOURS PMA (50 ng/ml)
18
12
6
0
0
0.1
1
5
10
Diphenyleneiodonium chloride [M]
20
50
HL-60 cells
Change in Mean Channel Fluorescence
20
DCF Fluorescence
EB Fluorescence
15
10
5
0
Passage 28
Passage 60
Phagocytosis
• Uptake of Fluorescent labeled particles
• Determination of intracellular or extracellular state of particles
How the assay works:
•
•
•
•
Particles or cells are labeled with a fluorescent probe
The cells and particles are mixed so phagocytosis takes place
The cells are mixed with a fluorescent absorber to remove fluorescence from membrane
bound particles
The remaining fluorescence
FITC-Labeled Bacteria
represents internal particles
Trypan Blue
FITC-Labeled Bacteria
pH Sensitive Indicators
Probe
Excitation
Emission
• SNARF-1
488
575
• BCECF
488
440/488
525/620
525
[2’,7’-bis-(carboxyethyl)-5,6-carboxyfluorescein]
Applications
• Probe Ratioing
– Calcium Flux (Indo-1)
– pH indicators (BCECF, SNARF)
Molecule-probe
Excitation
Emission
Calcium - Indo-1
Calcium- Fluo-3
Calcium - Fura-2
Calcium - Calcium Green
Magnesium - Mag-Indo-1
Phospholipase A- Acyl Pyrene
351 nm
488 nm
363 nm
488 nm
351 nm
351 nm
405, >460 nm
525 nm
>500 nm
515 nm
405, >460 nm
405, >460 nm
Ionic Flux Determinations
• Calcium
• Intracellular pH
Indo-1
BCECF
How the assay works:
• Fluorescent probes such as Indo-1 are able to bind to calcium in
a ratiometric manner
• The emission wavelength decreases as the probe binds available
calcium
1000
Ratio: intensity of 460nm / 405nm signals
0.8
0.7
800
0.6
600
0.4
400
0.3
200
0.2
0.1
Stimulation
0
RATIO [short/long]
0.5
0
36
72
108
Time (Seconds)
144
180
Flow Cytometry
0
0
Image Analysis
50
Time
(seconds)
100
150
200
Calcium ratios with Indo-1
1
1
2
2
3
Changes in the fluorescence were measured using
the Bio-Rad calcium ratioing software. The same
region in each wave length was measured and the
relative change in each region was recorded and
3
exported to a spread sheet for analysis..
460 nm
405/35 nm
Export data from measured regions to
Microsoft Excel
Export data from Excel data
base to Delta Graph
Ratio: intensity1 (460nm) / intensity2 (405/35nm)
0.8
0.7
0.6
cell 1
cell 2
cell 3
0.5
0.4
0.3
0.2
0.1
0
0
50
100
150
200
Probes for Ions
•
•
•
•
INDO-1
QUIN-2
Fluo-3
Fura -2
Ex350
Ex350
Ex488
Ex330/360
Em405/480
Em490
Em525
Em510
Membrane Potential
• Oxanol Probes
• Cyanine Probes
How the assay works:
Carbocyanine dyes released into the surrounding media as cells depolarize
Because flow cytometers measure the internal cell fluorescence, the kinetic changes
can be recorded as the re-distribution occurs
PMA Added
512
512
Green Fluorescence
Green Fluorescence
0
Repolarized Cells
1024
1024
fMLP Added
Depolarized Cells
0
0
•
•
1200
Time (sec)
2400
0
150
Time (sec)
300
Lipid Peroxidation
• Probe: 5 M cis-paranaric acid (Molecular Probes)
How the assay works:
• Cis-paranaric acid is a naturally fluorescent fatty acid which has
4 conjugated double bonds which become targeted by lipid
peroxidation reactions with a subsequent loss of fluorescence
Lipid Peroxidation
Paranaric Acid
1024
Over time - Paranaric acid loses its fluorescence as
the double bonds are destroyed
0
Cartoon of
the curve
that would
be derived
from the
data at left.
TIME (Seconds)
TIME (Seconds)
Data on left taken from Hedley, et al, Cytometry, 13: 686-692, 1992
“Caged” Photoactivatable Probes
Principle: Nitrophenyl blocking groups e.g. nitrophenyl ethyl ester
undergoes photolysis upon exposure to UV light at 340-350 nm
Available Probes
•
•
•
•
•
•
•
Ca++: Nitr-5
Ca++ - buffering: Diazo-2
IP3
cAMP
cGMP
ATP
ATP--S
Release of “Caged” Compounds
UV Beam
Culture dish
Release of “Cage”
Caged Nitric Oxide study
UV excited
Regions were selectively excited using UV
light to release the cage nitric oxide.
Images of the excited and adjacent control
region were then collected.
250
200
Fluorescence Emission at 515 nm
Control Region
150
Export data from measured
regions to Microsoft Excel
Export data from Excel data
dbase to Delta Graph
100
50
0
0
20
40
60
80
100
120
0
Time (seconds) after UV FLASH
140
160
FRAP
%F
Intense laser Beam
Bleaches Fluorescence
Time
Recovery of fluorescence
Zero time
10 seconds
30 seconds
Conclusions & Summary
Functional Studies In Cytometry
•
•
•
•
•
Oxygen radicals
Nitrogen radicals
Antioxidants
Cell viability
Organelle function
•
•
•
•
Lipid peroxidation
Membrane potential
Calcium fluxes
pH changes
Acknowledgements
•
•
•
•
•
•
•
•
•
Kathy Ragheb
Gretchen Lawler
Steve Kelley
Monica Shively
Dave Whittinghill
Stephanie Sincock
Karen Cornell
Karin Kooreman
Nian-Yu Li
Padma Narayanan (Smith Kline)
Wayne Carter (Pfizer)