BMS 631 - LECTURE 13 Flow Cytometry: Theory
Cell Function
J. Paul Robinson
SVM Professor of Cytomics &
Professor of Biomedical Engineering
Purdue University
Notice: The materials in this presentation are copyrighted
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Bindley Bioscience Center
Office: 765-494 0757
email; robinson@flowcyt.cyto.purdue.edu
WEB http://www.cyto.purdue.edu
7:17 PM
©1990-2013 J.Paul Robinson, Purdue University
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Cellular Response
•
•
•
•
•
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Cell death
Cell ‘suicide’
Ignore damage
Damage repair
Incorrect repair
©1990-2013 J.Paul Robinson, Purdue University
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Functional Assays
•intracellular pH
•intracellular calcium
•intracellular glutathione
•oxidative burst
•phagocytosis
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©1990-2013 J.Paul Robinson, Purdue University
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Oxidative Burst
•generation of toxic oxygen species
by phagocytic cells
•superoxide anion measured
with hydroethidine
•hydrogen peroxide measured with
2’,7’-dichlorofluorescin diacetate
(DCFH-DA)
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©1990-2013 J.Paul Robinson, Purdue University
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1000
Neutrophil Oxidative Burst
10
345
115
38
12
4
Unstimulated
Neutrophils
1
Scale
Log DCF
100
PMA-Stimulated
Neutrophils
0
600
1200
1800
2400
TIME (seconds)
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©1990-2013 J.Paul Robinson, Purdue University
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Phagocytosis
FITC-Labeled Bacteria
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©1990-2013 J.Paul Robinson, Purdue University
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Cellular Functions
• Cell Viability
• Phagocytosis
• Organelle Function
– mitochondria, ER
– endosomes, Golgi
• Oxidative Reactions
–
–
–
–
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Superoxide
Hydrogen Peroxide
Nitric Oxide
Glutathione levels
• Ionic Flux Determinations
–Calcium
–Intracellular pH
• Membrane Potential
• Membrane Polarization
• Lipid Peroxidation
©1990-2013 J.Paul Robinson, Purdue University
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Organelle Function
•
•
•
•
Mitochondria
Endosomes
Golgi
Endoplasmic Reticulum
Carbocyanine
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©1990-2013 J.Paul Robinson, Purdue University
Rhodamine 123
Ceramides
BODIPY-Ceramide
DiOC6(3)
Page 8
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 indicate for nitric oxide in a similar
manner to H2O2 so care must be used. DAF is a specific probe
available for Nitric Oxide
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©1990-2013 J.Paul Robinson, Purdue University
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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
DCF
OH-
Example: Neutrophil Oxidative Burst
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©1990-2013 J.Paul Robinson, Purdue University
Page 10
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
DCFH H O
2 2
Lymphocytes
counts
60
PMA-stimulated PMN
Control
40
20
DCF
0
.
1
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©1990-2013 J.Paul Robinson, Purdue University
1log
100
FITC 10
Fluorescence
1000
Page 11
Hydroethidine - Superoxide Production
15 minutes
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©1990-2013 J.Paul Robinson, Purdue University
45 minutes
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Endothelial Cell Oxidative Pathways
124.4
120
d
Percentage Change in
Mean Channel EB Fluorescence
140
100
118.9
100
d
94.3
a
80.9
a
80
60
c
52.5 58.9
b
be
40
57.7
50.2
b
e.g.
41.5 42.4
f
f
44.6
34.3
fg
h
20
0
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©1990-2013 J.Paul Robinson, Purdue University
Relative percentages of the
mean intracellular EB
fluorescence (O2-) in rat
pulmonary endothelial cells
(REC) 60 min after
stimulation with H2O2. This
figure is a summary of a
number of possible
oxidative pathways in
REC. The Y axis shows a
measurement of superoxide
anion via oxidation of
hydroethidine to ethidium
bromide, as a percentage of
the control (100%). XO
mediated pathways are
inhibited by nearly 50%. A
combination of inhibitors
of mitochondrial
respiration, as well as
solvents indicate the
baseline oxidation of the
probe (30-40%). (n=3)
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Oxidative Reactions
•
•
•
•
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Superoxide
Hydrogen Peroxide
Glutathione levels
Nitric Oxide
©1990-2013 J.Paul Robinson, Purdue University
Hydroethidine
Dichlorofluorescein
Monobromobimane
Dichlorofluorescein
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Calcium Flux
Flow Cytometry
Image Cytometry
Ratio: intensity of 460nm / 405nm signals
1000
0.8
0.7
800
0.6
600
0.5
400
0.4
0.3
200
0.2
0
Stimulation
0
36
72
108
144
Time (Seconds)
0.1
180
0
0
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©1990-2013 J.Paul Robinson, Purdue University
Time
50 (seconds)
100
150
200
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Membrane Potential
• Oxonol 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
fMLP Added
512
512
Green Fluorescence
Green Fluorescence
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Depolarized Cells
0
0
0
Repolarized Cells
1024
1024
PMA Added
1200
Time (sec)
2400
0
150
Time (sec)
©1990-2013 J.Paul Robinson, Purdue University
300
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Membrane Polarization
• Polarization/fluidity
Diphenylhexatriene
How the assay works: The DPH partitions into liphophilic portions of the cell
and is excited by a polarized UV light source. Polarized emissions are collected
and changes can be observed kinetically as cells are activated.
An image showing
DPH fluorescence in
cultured endothelial
cells.
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©1990-2013 J.Paul Robinson, Purdue University
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CD16 Expression on Normal Cultured PMN
negative
control
24 Hours
0 Hours
48 Hours
As cells age, the CD16 expression reduces
The “bright” CD16 antigen is lost first
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©1990-2013 J.Paul Robinson, Purdue University
Page 18
PI - Cell Viability
How the assay works:
• PI cannot normally cross the cell membrane
• If the PI penetrates the cell membrane, it is assumed
to be damaged
• Cells that are brightly fluorescent with the PI are
damaged or dead
Viable Cell
Damaged Cell
PI
PI
PI
PI
PI
PI
PI
PI
PI
PI
PI
PI
PI
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©1990-2013 J.Paul Robinson, Purdue University
PI
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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
Step 7C: Export data from Excel data
base to Delta Graph
1800
1600
1400
1200
1000
800
600
400
%change (DCF fluorescence)
Step 6C: Export data from measured
regions to Microsoft Excel
200
0
-200
cell 1
cell 2
cell 3
cell 4
cell 5
200 400
600 800 1000 1200 1400 1600 1800
Time in seconds
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©1990-2013 J.Paul Robinson, Purdue University
Page 20
Human Neutrophil
Phospolipase A2 activity
Leukotrienes
Lipid Peroxidation
OH.
Phagosome
H2O2
O2-
+
Stimulant
(PMA)
?
H2O2
SOD
O2
O2-
O2-
PKC
PCB
NADPH
Oxidase
SOD
GP
GR
GSH
NADPH + H+
Catalase
H2O2
GSSG
?
H2O + O2
+
H
NADP+
H2O
HMP
PCB
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PCB
(Reduced GSH level)
©1990-2013 J.Paul Robinson, Purdue University
Page 21
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
0.2
200
RATIO [short/long]
0.5
0.1
0
Stimulation
0
36
72
108
144
Time (Seconds)
180
Flow Cytometry
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0
0
50
Time
(seconds)
100
150
200
Image Analysis
©1990-2013 J.Paul Robinson, Purdue University
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Light Scatter Changes of PMN at 24 Hours
control
lps
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ar
bu
©1990-2013 J.Paul Robinson, Purdue University
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Phagocytosis
• Uptake of Fluorescent labeled particles
• Determination of intracellular or extracellular state of
particles
How the assay works:
•
•
•
•
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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
FITC-Labeled Bacteria
The remaining fluorescence
represents internal particles
©1990-2013 J.Paul Robinson, Purdue University
Page 24
Calcium ratioing study 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 exported to a spread sheet for analysis..
3
460 nm
405/35 nm
Export data from measured
regions to Microsoft Excel
0.8
Export data from Excel data
base to Delta Graph
0.5
0.4
Ratio: intensity1 (460nm) / intensity2 (405/35nm)
cell 1
cell 2
cell 3
0.7
0.6
0.3
0.2
0.1
0
0
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50
100
150
200
http://www.cyto.purdue.edu
©1990-2013 J.Paul Robinson, Purdue University
Page 25
On Calcofluor White
•
No warranties on this one but Calcofluor White M2R (Fluorescent brightener28, Sigma) may work but
staining may not be very specific. Lectins are another possible alternative.
A staining technique for differentiating starch granules and cell walls was developed for computerassisted studies of starchgranule distribution in cells of wheat (Triticum aestivum L.)caryopses. Blocks
of embedded caryopses were sectioned, exposing the endosperm tissue, and stained with iodine
potassium iodide (IKI) and Calcofluor White. Excessive tissue hydration during staining was avoided by
using stains prepared in 80% ethanol and using short staining times. The IKI quenched background
fluorescence which facilitated the use of higher concentrations of Calcofluor White. Cell wall definition
was improved with the IKI-Calcofluor staining combination compared to Calcofluor alone. The high
contrast between darkly stained starch granules and fluorescent cell walls permitted computer assisted
analysis of data from selected hard and soft wheat varieties. The ratio of starch granule area to cell
area was similar for both wheat classes. The starch granule sizes ranged from 2.1 microns 3 to 22,000
microns 3 with approximately 90% of the granules measuring less than 752 microns 3 (ca.11 microns
in diameter). Hard wheat samples had a greater number of small starch granules and a lower mean
starch granule area compared to the soft wheat varieties tested. The starch size distribution curve was
bimodal for both the hard and soft wheat varieties. Three-dimensional starch size distribution was
measured for four cells near the central cheek region of a single caryopsis. The percentage of small
granules was higher at the ends than at the mid-section of the cells
References: Biotech Histochem 1992 Mar;67(2):88-97
Block-surface staining for differentiation of starch and cell walls in wheat endosperm. Glenn GM, Pitts MJ, Liao K, Irving DW. Western Regional
Research Center, USDA-ARS, Albany, California 94710.
7:17 PM
Source:
From: Richard Haugland (richard.haugland@probes.com)
Date: Thu Feb 07 2002 - 17:04:48 EST
http://www.cyto.purdue.edu/hmarchiv/current/1041.htm
©1990-2013 J.Paul Robinson, Purdue University
Page 26
About SNARF-1
• SNARF®-1 carboxylic acid, acetate, succinimidyl ester
http://www.probes.com/servlets/product?region=Select&item=2
2801 is a very new probe that we have not yet tested for
assessing cell cycle.
• We have tested it for labeling cells and for cell tracing. I am not
aware of any publications that have used it for that, however.
However, it requires the same hydrolysis of the acetates as
does CFSE and has the same succinimidyl ester as CFSE and
should therefore have similar utility and have its fluorescence
decrease by half on cell division, as does CFSE.
• Its potential advantage is that it can be excited at 488 nm but
has red fluorescence so it may be complementary to CFSE.
7:17 PM
Source: From: Richard Haugland (richard.haugland@probes.com)
Date: Thu Jan 17 2002 - 20:10:07 EST
http://www.cyto.purdue.edu/hmarchiv/current/0872.htm
©1990-2013 J.Paul Robinson, Purdue University
Page 27
Summary
• There are a variety of functional probes
useful in flow cytometry
• Many require live cells for the entire
assay period
• Timing for kinetic assays is critical
• You must match the probe to the
excitation as usual
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©1990-2013 J.Paul Robinson, Purdue University
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