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Cytochrome c release, BAX translocation and apoptosis measurements

GG Georgia Greaves
MM Mateus Milani
MB Michael Butterworth
RC Rachel J. Carter
DB Dominic P. Byrne
PE Patrick A. Eyers
XL Xu Luo
GC Gerald M. Cohen
SV Shankar Varadarajan
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To quantitate cytochrome c release and BAX translocation, cells were grown on coverslips and treated with the specified inhibitors following a 0.5 h pre-treatment with the pan-caspase inhibitor Z-VAD.FMK (30 μM), and then fixed with 4% (v/v) paraformaldehyde and permeabilized with 0.5% (v/v) Triton X-100 in PBS, followed by incubation with primary and fluorophore-conjugated secondary antibodies prior to visualization under a fluorescent microscope. The extent of cytochrome c released from the mitochondria or BAX translocation to the mitochondria was quantified by counting at least 300 cells from three independent experiments. The extent of apoptosis in cells following different treatments was quantified using an Attune NxT flow cytometer (Thermo Fisher Scientific, Paisley, UK), as previously described. [24]

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