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After 20 days of culture, 14 bullfrogs were taken from each tank separately and executed by double destruction of medulla
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Superoxide measurement Cells were grown in a 12 well plate to about 50% confluence after which the media were removed and the cells were washed with HBSS (Hanks Balanced Salt Solution) followed by a 1 h pretreatment with 25 M dihydroethidium (DHE), from Life Technologies
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