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)

Superoxide (O
2
) – relatively long half life


Hydrogen Peroxide (H
2
O
2
)
Hydroxyl free radical (OH ) – very active oxidant

Increase in cellular ROS can cause oxidative damage to lipids, DNA , and protein

Electron Transport Chain: Complexes I-V


Significance of measuring of ROS

Evaluate oxidative stress status

Assess effectiveness of treatment

Challenging due to the instability of ROS

Currently available methods:

Lipid peroxidation assay


Electron spin resonance
Image analysis-based fluorescence microscopy

Flow cytometry


What is flow cyto metry ?


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


Hemorrhagic shock rat model

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

120
70
S1
Hemorrhagic Shock
1 hour
S2
S4
2 hours
S5
Stages of the Experiment


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.


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 37 0 C.

1 ml of MitoSOX Red solution was added to the blood sample and incubated in the dark for 30 minutes at 37 0 C


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.


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

MFI = 6782
Baseline
MFI = 4104
Shock
MFI = 9684
MFI = 3367
Treatment with CoQ10 Treatment without CoQ10

Mean Fluorescence Intensity of MitoSox Red at baseline, shock and after fluid resuscitation
Baseline
No CoQ10
5904 ± 245
CoQ10
5848 ± 327
Shock 6943 ± 210 6786 ± 502
Treatment 7992 ± 698 * 5469 ± 529

2000
1000
0
5000
4000
3000
9000
8000
7000
6000
Baseline
Shock Treatment
No CoQ10
CoQ10


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


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

Richard Hastings

Alicia Zeiger


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