Calcium Retention Capacity (CRC) Assay
Mitochondrial dysfunction is central to the pathogenesis of acute pancreatitis, ischemia-reperfusion injury, muscular dystrophies and neurodegeneration. Mitochondrial dysfunction is the result of a sudden increase in permeability of the inner mitochondrial membrane via the persistent opening of the mitochondrial permeability transition pore (mPTP), resulting in the disruption of ATP production and eventually cell death.
Figure 1: The core components of the mitochondrial PTP are thought to be cyclophilin D (CypD), the adenine nucleotide translocase (ANT) and the voltage-dependent anion channel (VDAC – also known as porin). IMM, inner mitrochondrial membrane, OMM, outer mitochondrial membrane, ROS, reactive oxygen species, CsA, cyclosporine A.
The mPTP opening and its inhibition can be evaluated by determining the calcium retention capacity1 (CRC) of mitochondria to Calcium Green™-5N, a low-affinity membrane impermeable dye that exhibits an increase in fluorescence emission intensity upon binding to calcium. Ten additions of 5μM calcium chloride is made to digitonin permeabilised cells at 5 min intervals, each eliciting a spike in signal that dissipates due to the uptake of calcium from the matrix. Opening of the mPTP collapses the mitochondrial membrane potential, which results in release of the the accumulated calcium from the matrix, causing a sudden and sustained increase in fluorescence signal.
- Potential mitochondrial pore inhibitors or inducers can be assayed over a 7-point concentration range to generate an EC50 or as single concentrations for library screening
- CRC assay runs in 96 well format utilizing a FLIPR TETRA
- CRC assay runs in either permeabilised HepG2 (human hepatocytoma) or SH-SY5Y (human neuroblastoma) cells
- Quick turnaround times of 2-4 weeks depending on project type
