Transcript Measuring Leukocyte Mitochondrial Superoxide Using Flow Cytometry

Reactive Oxygen Species (ROS)
 Highly reactive molecules containing oxygen and
unpaired electrons, which are byproducts of
oxidative phosphorylation in mitochondria
 Three major types of reactive oxygen species
(ROS)
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Superoxide (O2-) – relatively long half life
Hydrogen Peroxide (H2O2)
Hydroxyl free radical (OH-) – very active oxidant
 Increase in cellular ROS can cause oxidative
damage to lipids, DNA, and protein
Mitochondrion and ROS
Electron Transport Chain: Complexes I-V
Measurement of ROS
 Significance of measuring of ROS
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Evaluate oxidative stress status
Assess effectiveness of treatment
 Challenging due to the instability of ROS
 Currently available methods:
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Lipid peroxidation assay
Electron spin resonance
Image analysis-based fluorescence microscopy
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Flow cytometry
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Flow Cytometry
 What is flow cytometry?
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Flow = cells are in motion
Cyto = cells
Metry = measure
Flow cytometry = measuring characteristics/
properties of cells while they are in a fluid stream
 Laser-based, biophysical technology
 Can simultaneously analyze multiple physical
and/or chemical characteristics of thousands of
cells per second
Mechanism of Flow Cytometry
BD LSRII Flow Cytometer
Aim
Methods
 Hemorrhagic shock rat model
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Removal of 40% of blood volume from
anesthetized rats over one hour
Fluid resuscitation with/without Coenzyme Q10
 Coenzyme Q10 is a key component in the
electron transport chain in mitochondria and
plays roles in ATP production and
scavenging ROS
Hypoperfusion and reperfusion injury cause
increases in ROS production
Experimental Design
Fluid Resuscitation
S3
MAP (mmHg)
120
S4
70
Hemorrhagic Shock
1 hour
S1
2 hours
S2
Stages of the Experiment
S5
Reagents
 Monoclonal antibodies (CD45) – leukocytes
 MitoSOX Red (Molecular Probes, Invitrogen)
A novel fluorogenic dye
 Highly selective detection of superoxide in
the mitochondria of live cells
 Exhibits red fluorescence when oxidized
by superoxide.
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Methods
 Blood samples (0.1 ml) were collected at
baseline, shock, and after fluid
resuscitation with or without intravenous
Coenzyme Q10.
 Unstained and single stained controls
were obtained at baseline.
 20 µl of blood were incubated with 2 µl
CD45 in 200 µl of phosphate buffered
saline (PBS) for 5 minutes at 370C.
 1 ml of MitoSOX Red solution was added
to the blood sample and incubated in the
dark for 30 minutes at 370C
Methods
 1 ml of PBS was added
after incubation and
then centrifuged at 1000
rpm for 5 minutes
 Supernatant was
disregarded and the
pellet cells were then
suspended in 1 ml of
PBS.
Methods
 Three analyses with 10,000 leukocytes
(CD45 positive cells) were conducted using flow
cytometry (Becton Dickinson LSRII) for each
sampling period
 Mean fluorescence intensity (MFI) of MitoSOX
Red was obtained
Data Acquisition and Analysis
MFI = 6782
MFI = 4104
Baseline
Shock
MFI = 3367
Treatment with CoQ10
MFI = 9684
Treatment without CoQ10
Results
Mean Fluorescence Intensity of MitoSox Red at
baseline, shock and after fluid resuscitation
No CoQ10
CoQ10
Baseline
5904 ± 245
5848 ± 327
Shock
6943 ± 210
6786 ± 502
Treatment
7992 ± 698 *
5469 ± 529
Results
Mean Fluorescence Intensity of MitoSOX
Red
9000
8000
7000
6000
5000
No CoQ10
4000
CoQ10
3000
2000
1000
0
Baseline
Shock
Treatment
Lessons Learned
 Sample preparation in the dark !!
MitoSOX Red is very sensitive
to light exposure
 Wash cells is an important step to
remove excessive unbound
monoclonal antibodies to avoid
background fluorescence
 Running samples on time
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Conclusions
Research Team
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Dr. Janet Pierce, PI
Dr. Richard Clancy, grant consultant
Paul Bennetts, doctoral student
Amanda Thimmesch, Research Associate
Flow Cytometry Center, University of
Kansas Medical Center
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Richard Hastings
Alicia Zeiger
Acknowledgement
 This research was sponsored by the
TriService Nursing Research Program,
Uniformed Services University of the Health
Sciences (HU0001-11-1-TS09).
 This presentation was supported partially by
the Postdoc Travel Scholarship funded by the
KU Hospital Auxiliary and Faculty Travel
Fund by the Office of Grant and Research
(OGR), School of Nursing, University of
Kansas