Template:SUIT text D008

SUIT-010 O2 ce-pce D008 is designed for the evaluation of optimum digitonin concentration for selective cell membrane permeabilization, a requirement to account for differences between cell types, the concentration of cells, and variability between batches of digitonin. After inhibition of the endogenous ROUTINE respiration by rotenone, respiration of intact cells is not stimulated by succinate and ADP. Subsequent stepwise digitonin titration yields gradual permeabilization of the cell membrane, indicating by the increase of respiration up to full permeabilization. Respiration is constant within optimal digitonin concentration and inhibited by over-titration with digitonin, since the mitochondrial outer membrane is affected and cytochrome c can be released. Therefore, it is recommended to titrate cytochrome c after the optimal digitonin concentration has been reached. In other SUIT protocols for permeabilized cells, the respiration of pce is stable, if the optimal digitonin concentration is used.