2. Reactive Oxygen Species (ROS)

3. Mitochondrion and ROS Electron Transport Chain: Complexes I-V

4. Measurement of ROS  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

5. Flow Cytometry  What is flow cytometry?  = cells are in motion  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

6. Mechanism of Flow Cytometry

7. BD LSRII Flow Cytometer

8. Aim

9. Methods

10. Experimental Design Stages of the Experiment Stages of the Experiment S5 S1 S2 2 hours S4 Hemorrhagic Shock 1 hour S3 120 70 MAP (mmHg) Fluid Resuscitation

11. 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 the mitochondria of live cells  Exhibits red fluorescence when oxidized by superoxide. by superoxide.

12. 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 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

13. 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.

14. 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

15. Data Acquisition and Analysis

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

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

18. Results

19. 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

20. Conclusions

21. Research Team  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

22. 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