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