Functiona l assays - Principle s and Methods J Paul Robinson Purdue University Cytometry Laboratories These slides are on the PURDUE CYTOMETRY WEB SITE 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
43
Embed
Functional assays - Principles and Methods J Paul Robinson Purdue University Cytometry Laboratories These slides are on the PURDUE CYTOMETRY WEB SITE .
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Functional assays - Principles and Methods
J Paul Robinson
Pur
due
Uni
vers
ity C
ytom
etry
Lab
orat
orie
s
These slides are on the PURDUE CYTOMETRY WEB SITEThese slides are on the PURDUE CYTOMETRY WEB SITE
http://flowcyt.cyto.purdue.edu
These slides are on the PURDUE CYTOMETRY WEB SITEThese 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 center-left is Gdansk.
Warsaw
Gdansk
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
KineticsPrinciple 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
Flu
ores
cenc
e
Fluorescent IndicatorsHow 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 Rhodamine 123
• Endosomes Ceramides
• Golgi BODIPY-Ceramide
• Endoplasmic Reticulum DiOC6(3)
Carbocyanine
DCFH-DA DCFH DCFDCF
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’-dichlorofluoresceinCellular Esterases
H2O2
DCFH-DA
DCFH-DADCFH-DA
DCFHDCFH
DCF
H OH O 2 22 2
Lymphocytes
Monocytes
Neutrophils
log FITC Fluorescence.1 1000 100 10 1
0
20
40
60
cou
nts
PMA-stimulated PMNControl
80
HydroethidineHE 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
Both these images are cells stained to measure for H2O2 production.
Some examples of rapidly changing antigen expression systems
BACTERIALINFECTION
TNFIL-1
a
b cd
Endothelial Cells
E-selectin & P-selectin
ICAM-1
L-selectin &CHO ligands(e.g. sLex)
CD11b
Neutrophils
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, L-selectin 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).
E-selectin
ICAM-1
H2O2
O2-
OH-
NO.
NFB
TNF
VCAM
ROS
Bradykinin
NO.
? (NOO-)
-
+ +
+
++
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.
P-selectin
CD11b
membrane damage conjugated dienes
+ stimulatory effect- inhibitory effect
++
-
++
Oxidative Reactions
• Superoxide Hydroethidine
• Hydrogen Peroxide Dichlorofluorescein
• Glutathione levels Monobromobimane
• Nitric Oxide Dichlorofluorescein
Rat Pulmonary Artery Endothelial CellsOxidization via H2O2
Periodicity of Fluorescence
Purdue University Cytometry Laboratories
Meridian UltimaTM Analysis
Macrovascular Endothelial Cells in Culture
Time (minutes)0 60
Confocal System
Culture System
Step 1: Cell Culture
Step 2: Cell Wash
Lab-Tek
1 2
3 4
5 6
7 8
top view
side view
170 M coverslip
Step 3: Transfer to Lab-Tek plates
confocal microscopeoil immersionobjective
37o heated stage
stimulant/inhibitor added
Step 4: Addition of DCFH-DA, Indo-1, or HE
Hydrogen peroxide measurements with DCFH-DA
0
200
400
600
800
1000
1200
1400
1600
1800
2000
0 500 1000 1500 2000 2500 3000Time in seconds
cell 1
cell 2
cell 3
cell 4
cell 5
% c
hang
e (D
CF
fluo
resc
ence
)
525 nm
1 2
3
45
Step 6B: Export data from measured regions to Microsoft Excel
Step 7B: Export data from Excel data base to Delta Graph
Change in fluorescence was measured using Bio-Rad software and the data exported to a spread sheet for analysis.
Superoxide measured with hydroethidine
Export data from Excel data
base to Delta Graph
Export data from measuredregions 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.
%ch
ange
(D
CF
fluo
resc
ence
)
-200
0200
400600800
100012001400
16001800
cell 1
cell 2
cell 3
cell 4
cell 5
Time in seconds
1000 1200 1400 1600 1800600 800 200 400
Promyelocyte
NeutrophilMyelocyte
Metamyelocyte
Padma Narayanan figures hl60.ppt
0
6
12
18
24
30
36
0 HOURS 24 HOURS 48 HOURS 72 HOURS 96 HOURS
Ch
ange
in M
ean
DC
F F
luor
esce
nce
0 ng/ml PMA
8 ng/ml PMA
50 ng/ml PMA
a
ab
b1
c1
c
d
ed1
e1
0
6
12
18
24
30
36
0 0.1 1 5 10 20 50
Diphenyleneiodonium chloride [M]
0 HOURS PMA (8 ng/ml)
0 HOURS PMA (50 ng/ml)
96 HOURS PMA (8 ng/ml)
96 HOURS PMA (50 ng/ml)
Ch
ange
in M
ean
DC
F F
luor
esce
nce
DCF Fluorescence
EB Fluorescence
0
5
10
15
20
Ch
ange
in M
ean
Ch
ann
el F
luor
esce
nce
Passage 28 Passage 60
HL-60 cells
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
• 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
1
2
33
2
1
405/35 nm460 nm
Calcium ratios 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