Saponin and digitonin both can permeabilize cells. However, based on many respiration assay protocols I read, saponin is used for tissue permeabilization and digitonin is used for cell permeabilization. What are the differences between these two in terms of their permeability? Can they be used interchangeably for cultured cell respiration assay?
Dear Yiming,
As you correctly mentioned, "at low concentrations, digitonin or saponin do permeabilize the plasma membranes completely and selectively due to their high cholesterol content, whereas mitochondrial membranes with lower cholesterol content are affected only at higher concentrations." Reference: (PDF) High-Resolution Respirometry: OXPHOS Protocols for Human Cells and Permeabilized Fibers from Small Biopsies of Human Muscle.
Thus, I think you can try to set your protocol using the detergent you get the best respiratory coupling with. In my experience, digitonin is a milder detergent than saponin, which would be my first option to permeabilize cultured cells. However, if you don't have it, and want to try Saponin, you should titrate it and find a concentration in which you achieve correct permeabilization and the mitochondrial respiratory control is not lost; i.e. a higher respiratory rate (state III) after addition of ADP+Pi. In any case, the concentrations of all detergents have to be titrated prior to the experiments, since the purity and effectiveness do vary depending on brands, batches and formulation.
Best,
Alfredo
Dear Yiming,
As you correctly mentioned, "at low concentrations, digitonin or saponin do permeabilize the plasma membranes completely and selectively due to their high cholesterol content, whereas mitochondrial membranes with lower cholesterol content are affected only at higher concentrations." Reference: (PDF) High-Resolution Respirometry: OXPHOS Protocols for Human Cells and Permeabilized Fibers from Small Biopsies of Human Muscle.
Thus, I think you can try to set your protocol using the detergent you get the best respiratory coupling with. In my experience, digitonin is a milder detergent than saponin, which would be my first option to permeabilize cultured cells. However, if you don't have it, and want to try Saponin, you should titrate it and find a concentration in which you achieve correct permeabilization and the mitochondrial respiratory control is not lost; i.e. a higher respiratory rate (state III) after addition of ADP+Pi. In any case, the concentrations of all detergents have to be titrated prior to the experiments, since the purity and effectiveness do vary depending on brands, batches and formulation.
Best,
Alfredo
Alfredo Cabrera-Orefice Thank you, Alfredo. I don't have digitonin in hand, so I will try to optimize using saponin as you suggested. Really appreciate it.
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