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Mitochondrial swelling assay (PTP opening) - (Jul/05/2013 )


Anyone here with experience with the mitochondrial swelling assay for the measurement of the opening of the permeability transition pore? I'm using the method that evaluates the decrease in absorbance at 540 nm. I'm working with a fungus and there's nothing in the literature about this. I get a "weird" profile (see attachment).

1) the "temporal dynamics" are quite different from what is observed in mammalian cells (normally I see 5 minute experiments and in my case I observed decrease in absorbance at least during the first half hour)

2) I get a very weird (but very reproducible) peak at 2-5mins after the addition of CaCl2. This is not inhibited by cyclosporin A, so I don't know what this is... any idea??

3) I get a decrease in absorbance after this, prolonged in time that is inhibited by cyclosporin A, so I guess this is the PTP opening.

Can someone please comment on this?

Thank you.

Attached Image


C'mon, help me! :)