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Page 1©1990-2013 J.Paul Robinson, Purdue University09:36 PM
BMS 631 - LECTURE 13 Flow Cytometry: Theory
Bindley Bioscience CenterOffice: 765-494 0757email; [email protected]
Cell Function
J. Paul RobinsonSVM Professor of Cytomics &Professor of Biomedical EngineeringPurdue University
WEB http://www.cyto.purdue.edu
Notice: The materials in this presentation are copyrighted materials. If you want to use any of these slides, you may do so if you credit each slide with the author’s name. It is illegal to copy these to CourseHero or any other online theft system.
Page 2©1990-2013 J.Paul Robinson, Purdue University09:36 PM
Cellular Response
• Cell death• Cell ‘suicide’• Ignore damage• Damage repair• Incorrect repair
Page 3©1990-2013 J.Paul Robinson, Purdue University09:36 PM
Functional Assays
•intracellular pH•intracellular calcium•intracellular glutathione•oxidative burst•phagocytosis
Page 4©1990-2013 J.Paul Robinson, Purdue University09:36 PM
Oxidative Burst
•generation of toxic oxygen speciesby phagocytic cells
•superoxide anion measuredwith hydroethidine
•hydrogen peroxide measured with2’,7’-dichlorofluorescin diacetate(DCFH-DA)
Page 5©1990-2013 J.Paul Robinson, Purdue University09:36 PM
TIME (seconds)
2400 1800 1200 600
Scale
34511538
124
Neutrophil Oxidative Burst
PMA-StimulatedNeutrophils
UnstimulatedNeutrophils
0
Log
DC
F1
1010
010
00
Page 7©1990-2013 J.Paul Robinson, Purdue University09:36 PM
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
Page 8©1990-2013 J.Paul Robinson, Purdue University09:36 PM
Organelle Function
• Mitochondria Rhodamine 123• Endosomes Ceramides• Golgi BODIPY-Ceramide
• Endoplasmic Reticulum DiOC6(3)
Carbocyanine
Page 9©1990-2013 J.Paul Robinson, Purdue University09:36 PM
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
Page 10©1990-2013 J.Paul Robinson, Purdue University09:36 PM
Hydroethidine
HE EB
NCH2CH3
NH2H2N
H Br-NCH2CH3
NH2H2N
+
O2-
Phagocytic Vacuole
SODH2O2
NADPH
NADP
O2
NADPH Oxidase
OH-
O2-
DCFDCF
HE
OO22--
HH22OO22
DCFDCF
Example: Neutrophil Oxidative Burst
Page 11©1990-2013 J.Paul Robinson, Purdue University09:36 PM
DCFH-DA DCFH DCF
COOHH
Cl
O
O-C-CH3
O
CH3-C-O
Cl
O
COOHH
Cl
OHHO
Cl
O
COOHH
Cl
OHO
Cl
O
Fluorescent
Hydrolysis
Oxidation
2’,7’-dichlorofluorescin
2’,7’-dichlorofluorescin diacetate
2’,7’-dichlorofluorescein
Cellular Esterases
H2O2
DCFH-DA
DCFH-DADCFH-DA
DCFHDCFH
DCF
H OH O 2 22 2Lymphocytes
Monocytes
Neutrophils
log FITC Fluorescence.1
1000
100 10
1
0
20
40
60
cou
nts
PMA-stimulated PMNControl
80
Page 12©1990-2013 J.Paul Robinson, Purdue University09:36 PM
Hydroethidine - Superoxide Production
15 minutes 45 minutes
Page 13©1990-2013 J.Paul Robinson, Purdue University09:36 PM
Endothelial Cell Oxidative Pathways
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)
100
52.5 58.9
94.3
57.7
80.9
124.4118.9
50.241.5 42.4 44.6
34.3
0
20
40
60
80
100
120
140
aa
be
c
b b
dd
e.g.
f f fg
h
Mea
n C
han
nel
EB
Flu
ore
scen
ceP
erce
nta
ge
Ch
ang
e in
EC only
EC + 5mM A
llopurin
ol & 1mM
DM
SO
ECEC + 1mM
cyan
ide & 1mM
DM
SO
EC + 1mM C
yanide
EC + 1mM A
zide
EC + 1mM D
eferoxam
ine
EC + 10mM M
annito
l
EC + 8mM B
enzo
ate
EC + 1mM D
MSO
EC + 5mM D
PI
+ 5mM D
PI
EC + 5mM O
xypurinol
EC + 5mM A
llopurin
ol
+ 5mM A
llopurin
ol
EC + 5mM A
llopurin
ol & 1mM
Cn
Page 14©1990-2013 J.Paul Robinson, Purdue University09:36 PM
Oxidative Reactions
• Superoxide Hydroethidine• Hydrogen Peroxide Dichlorofluorescein• Glutathione levels Monobromobimane• Nitric Oxide Dichlorofluorescein
Page 15©1990-2013 J.Paul Robinson, Purdue University09:36 PM
Calcium Flux
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 50 100 150 200
Rat
io: in
tens
ity o
f 46
0nm
/ 4
05nm
sig
nals
Time (seconds)
Time (Seconds)0 36 72 108 144 180
RAT
IO [s
hort/
long
]0
200
400
600
800
1000
StimulationStimulation
Flow Cytometry Image Cytometry
Page 16©1990-2013 J.Paul Robinson, Purdue University09:36 PM
Membrane Potential
• Oxonol Probes • Cyanine ProbesHow 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
Time (sec)
Gre
en F
luor
esce
nce
Repolarized Cells
051
210
24
0 300 1500 1200 2400Time (sec)
051
210
24G
reen
Flu
ores
cenc
e
PMA Added fMLP Added
Depolarized Cells
Page 17©1990-2013 J.Paul Robinson, Purdue University09:36 PM
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.
Page 18©1990-2013 J.Paul Robinson, Purdue University09:36 PM
CD16 Expression on Normal Cultured PMN
0 Hours
negativecontrol
48 Hours
24 Hours
As cells age, the CD16 expression reducesThe “bright” CD16 antigen is lost first
Page 19©1990-2013 J.Paul Robinson, Purdue University09:36 PM
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
PIPI
PIPI
PIPI
PIPI
PIPI
PIPI
PIPI
PIPIPIPI
PIPI
PI
PIPI
PIPI
PIPI
Viable Cell Damaged Cell
Page 20©1990-2013 J.Paul Robinson, Purdue University09:36 PM
Superoxide measured with hydroethidine
Step 7C: Export data from Excel data base to Delta Graph
Step 6C: Export data from measured regions to Microsoft Excel
cell 1
cell 2
cell 3cell 4
cell 5
Change in fluorescence was measured using Bio-Rad software and the data exported to a spread sheet for analysis.
-2000
200
400600800
10001200140016001800
cell 1
cell 2
cell 3
cell 4
cell 5
Time in seconds1000 1200 1400 1600 1800600 800 200 400%
chan
ge (
DC
F fl
uore
scen
ce)
Page 21©1990-2013 J.Paul Robinson, Purdue University09:36 PM
Phagosome
O2
O2-
H2O2
NADPH + H+
NADP+
HMP
NADPH
Oxidase
GSSG
GSH
GRGP
H2O2SOD
O2-
H+
H2O
Catalase
H2O + O2
PCB
SOD
PCB(Reduced GSH level)
Stimulant
PKC
PCB
(PMA)
Human Neutrophil
?
?+
O2-
OH.
Lipid Peroxidation
Phospolipase A2 activity
Leukotrienes
H2O2
Page 22©1990-2013 J.Paul Robinson, Purdue University09:36 PM
Ionic Flux Determinations
• Calcium Indo-1
• Intracellular pH BCECFHow 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
Time (Seconds)0 36 72 108 144 180
RAT
IO [s
hort/
long
]0
200
400
600
800
1000
StimulationStimulation0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 50 100 150 200
Rat
io: i
nten
sity
of 4
60nm
/ 40
5nm
sig
nals
Time (seconds)
Flow Cytometry Image Analysis
Page 23©1990-2013 J.Paul Robinson, Purdue University09:36 PM
Light Scatter Changes of PMN at 24 Hours
control
lps bu
ar
Page 24©1990-2013 J.Paul Robinson, Purdue University09:36 PM
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
represents internal particles
FITC-Labeled Bacteria
Page 25©1990-2013 J.Paul Robinson, Purdue University09:36 PM
1
2
33
2
1
405/35 nm460 nm
Calcium ratioing study with Indo-1
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..
Export data from measured regions to Microsoft Excel
Export data from Excel data base to Delta Graph
50 100 150 2000
0.1
0.20.3
0.40.5
0.60.7
0.8
0
cell 1 cell 2 cell 3
Ratio: intensity1 (460nm) / intensity2 (405/35nm)
http://www.cyto.purdue.edu
Page 26©1990-2013 J.Paul Robinson, Purdue University09:36 PM
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 computer-assisted 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
Source:From: Richard Haugland ([email protected])Date: Thu Feb 07 2002 - 17:04:48 EST http://www.cyto.purdue.edu/hmarchiv/current/1041.htm
References: Biotech Histochem 1992 Mar;67(2):88-97Block-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.
Page 27©1990-2013 J.Paul Robinson, Purdue University09:36 PM
About SNARF-1
• SNARF®-1 carboxylic acid, acetate, succinimidyl ester http://www.probes.com/servlets/product?region=Select&item=22801 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.
Source: From: Richard Haugland ([email protected])Date: Thu Jan 17 2002 - 20:10:07 EST http://www.cyto.purdue.edu/hmarchiv/current/0872.htm