Methods for the assessment of mitochondrial membrane permeabilization in apoptosis

Abstract

Mitochondrial membrane permeabilization (MMP) is considered as the "point-of-no-return" in numerous models of programmed cell death. Indeed, mitochondria determine the intrinsic pathway of apoptosis, and play a major role in the extrinsic route as well. MMP affects the inner and outer mitochondrial membranes (IM and OM, respectively) to a variable degree. OM permeabilization culminates in the release of proteins that normally are confined in the mitochondrial intermembrane space (IMS), including caspase activators (e.g. cytochrome c) and caspase-independent death effectors (e.g. apoptosis-inducing factor). Partial IM permeabilization disrupts mitochondrial ion and volume homeostasis and dissipates the mitochondrial transmembrane potential ( m ). The assessment of early mitochondrial alterations allows for the identification of cells that are committed to die but have not displayed yet the apoptotic phenotype. Several

Role of the c subunit of the FOATP synthase in mitochondrial permeability transition

“…To monitor the opening of the PTPC in living cells, we took advantage of the calcein-Co 2+ assay. 53,54 In this setting, cells are loaded with an esterified form of calcein, which is retained within the plasma membrane (thanks to the activity of cytoplasmic esterases) and freely diffuses into all subcellular compartments (including mitochondria), followed by bivalent cobalt (Co 2+ ) ions. Co 2+ ions quench the calcein-dependent cytoplasmic and nuclear, but not mitochondrial, fluorescence, as they are excluded from the mitochondrial matrix owing to the barrier function of the inner mitochondrial membrane.…”

“…Thus, the induction of MPT can be followed in cellula as a further drop in calcein-dependent fluorescence. 53,54 HeLa cells transfected with a control siRNA (or mock-transfected) and loaded with calcein-Co 2+ responded to the administration of 1 μM ionomycin (a ionophore that triggers MPT by promoting a mitochondrial Ca 2+ overload) with a rapid decrease in calcein fluorescence. Such a drop could be fully prevented by the pre-administration of cyclosporine a (CsA, a chemical inhibitor of CYPD), confirming that it resulted from the opening of the PTPC ( Fig.…”

Molecular mechanisms of ATP secretion during immunogenic cell death

“…Apoptotic cell death was monitored by virtue of PS exposure and the uptake of the vital dye DAPI. 59,60 To this aim, cells were washed twice with PBS and resuspended in 1 Â binding buffer supplemented with PE-or fluorescein isothiocyanate (FITC)-conjugated AnnexinV (BD Biosciences, Franklin Lakes, NJ, USA) and DAPI, as per manufacturer's instructions. For the assessment of plasma membrane permeability, cells were incubated in culture medium supplemented with 1 mM YO-PRO-1 (Molecular Probes-Life Technologies, Eugene, CA, USA) for 30 min at 37 1C.…”

Measuring apoptosis at the single cell level

“…There are other dyes available that also measure Δ Ψm each with their own advantages and drawbacks (12). Rhodamine 123 is similar to TMRE but quenches at high concentrations and is also quite toxic to most cells.…”