Bone Abstracts

Caspases belong to cysteine proteases participating in apoptosis and inflammation. However, there is recent evidence about their functions also in other events such as cell differentiation. This is supported also by our latest research of odontogenesis and osteogenesis. With the increasing interest in caspases due to their apoptosis-related therapies but also emerging non-apoptotic roles, exact evaluation of their impact at single cell level becomes challenging.

We have recently reported about caspase-3 detection at femtogram level (10⁻¹⁵ g) in cell populations of embryonic micromasses of chondrogenic mesenchymal cells. To evaluate active caspase-3 in individual cells, a miniaturized device was developed.

Such miniaturized device enables quantitation of caspase-3 just in single apoptotic and non-apoptotic cell. The detection is based on the specific cleavage of modified luciferin by caspase-3, bioluminescent emissions of photons and their detection by photomultiplier tube working in the photon counting regime. Mouse front limbs at the digitalization stage were examined. Digital vs intedigital cells were used to verify the novel method. Caspase-3 in these cells was simultaneously evaluated by immunohistochemistry and flow cytometry.

Mass of caspase-3 per cell in non-apoptotic cells was under detection limit, whereas the mass in interdigital apoptotic cells was around 5–6 fg corresponding to 10⁵ of molecules. These masses of caspase-3 in mouse apoptotic cells are in a good agreement with the data published so far. Recently, further precision of instrumentation is under process using a low volume technology to quantify the natural content of active caspase-3 in nonapoptotic mouse cells.