A significant proportion of environmental oxygen is metabolized by single electron reductions in living cells. This type of univalent reduction creates highly reactive free radical species which can irreversibly damage cellular constituents, eventuating in cellular injury and death. Oxygen radicals have been implicated in several pathologic processes, including myocardial re-oxygenation injury, various types of chemotoxicity, carcinogenesis, aging and radiation injury. One in vitro system particularly suitable for the study of these processes utilizes cultured neonatal mouse heart cells. The balance of free radical species within these cells may be altered by straightforward experimental manipulations to simulate various environmental stresses associated with free radical production. Free radical damage is largely directed against the lipid constituents of the cell membrane, which are chemically accessible to radicals produced within both the intracellular and extracellular spaces. Thus, a parameter quantitating membrane integrity provides a sensitive means of determining the extent of radical-mediated injury. We have developed and previously reported such a method using fluorescent-tagged antimyosin and a laser-activated cell sorting system (FACS III). Using this system in conjunction with other established methods, the mechanism and extent of radical-mediated cellular damage can be assessed in vitro. Various techniques of minimizing this injury can be easily and quantitatively assessed using these methods.
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