Optimal Percoll concentration facilitates flow cytometric analysis for annexin V/propidium iodine-stained ischemic brain tissues

Authors

  • Wei-Sheng Juan,

    1. Neurophysiology Laboratory, Neurosurgical Service, Department of Surgery, National Cheng Kung University Medical College and Hospital, Tainan, Taiwan
    2. Department of Anesthesiology, Buddhist Tzu-Chi University and Buddhist Tzu Chi General Hospital, Hualien, Taiwan
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    • Wei-Sheng Juan and Hsiao-Wen Lin contributed equally to the work.

  • Hsiao-Wen Lin,

    1. Neurophysiology Laboratory, Neurosurgical Service, Department of Surgery, National Cheng Kung University Medical College and Hospital, Tainan, Taiwan
    2. Department of Anesthesiology, Buddhist Tzu-Chi University and Buddhist Tzu Chi General Hospital, Hualien, Taiwan
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  • Ying-Hsin Chen,

    1. Neurophysiology Laboratory, Neurosurgical Service, Department of Surgery, National Cheng Kung University Medical College and Hospital, Tainan, Taiwan
    2. Department of Anesthesiology, Buddhist Tzu-Chi University and Buddhist Tzu Chi General Hospital, Hualien, Taiwan
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  • Hung-Yi Chen,

    1. Institute of Pharmacy, China Medical University, Taichung, Taiwan
    2. Department of Anesthesiology, Buddhist Tzu-Chi University and Buddhist Tzu Chi General Hospital, Hualien, Taiwan
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  • Yu-Chang Hung,

    1. Neurophysiology Laboratory, Neurosurgical Service, Department of Surgery, National Cheng Kung University Medical College and Hospital, Tainan, Taiwan
    2. Department of Anesthesiology, Buddhist Tzu-Chi University and Buddhist Tzu Chi General Hospital, Hualien, Taiwan
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  • Shih-Huang Tai,

    1. Neurophysiology Laboratory, Neurosurgical Service, Department of Surgery, National Cheng Kung University Medical College and Hospital, Tainan, Taiwan
    2. Department of Anesthesiology, Buddhist Tzu-Chi University and Buddhist Tzu Chi General Hospital, Hualien, Taiwan
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  • Sheng-Yang Huang,

    1. Neurophysiology Laboratory, Neurosurgical Service, Department of Surgery, National Cheng Kung University Medical College and Hospital, Tainan, Taiwan
    2. Department of Anesthesiology, Buddhist Tzu-Chi University and Buddhist Tzu Chi General Hospital, Hualien, Taiwan
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  • Tsung-Ying Chen,

    1. Institute of Pharmacy, China Medical University, Taichung, Taiwan
    2. Department of Anesthesiology, Buddhist Tzu-Chi University and Buddhist Tzu Chi General Hospital, Hualien, Taiwan
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  • E-Jian Lee

    Corresponding author
    1. Neurophysiology Laboratory, Neurosurgical Service, Department of Surgery, National Cheng Kung University Medical College and Hospital, Tainan, Taiwan
    2. Department of Anesthesiology, Buddhist Tzu-Chi University and Buddhist Tzu Chi General Hospital, Hualien, Taiwan
    • Department of Surgery, National Cheng Kung University Medical Center and Medical School, 138 Sheng-Li Road, Tainan 70428, Taiwan
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Abstract

We sought to determine the optimal Percoll concentration for ischemic rat brain prepared for flow cytometric (FC) measurements. Animals were subjected to the right middle cerebral artery (MCA) occlusion, and were euthanized at 3, 12, 24, and 72 h after reperfusion onset. The brains were processed by different concentrations (unisolated, 20, 25, 30, or 40%) of Percoll and stained with annexin V/propidium iodine (PI). Ischemic brain damage was evaluated by FC analysis and image analysis for histologic sections. The relative susceptibility of different phenotypes of cells to necrotic and apoptotic damage were evaluated by the FC analyses for the immunohistochemistry, PI, and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-processed brain tissues. Our results showed that FC analysis effectively detected the extent and maturation of apoptotic/necrotic brain damage, and the results were consistent with those determined from histologic brain sections. Neuron was more vulnerable to apoptosis than glia, whereas both cellular phenotypes were compatible in susceptibility for necrotic cell death. Percoll at a low concentration (20%) could effectively remove tissue debris without affecting membranous integrity of the injured neurons. Conversely, high percentages of Percoll (30–40%) substantially increased membranous damage for the injured cells. These results supported the application of FC to determine the extent and progression in time, as well as relative phenotypes of apoptotic/necrotic cell deaths following ischemic damage. We highlighted the use of Percoll at low percentages to facilitate the removal of tissue debris and to improve membrane integrity preservation for the injured neurons. © 2012 International Society for Advancement of Cytometry.

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