Background: In vitro studies of osteoblasts traditionally use Alizarin Red as a golden standard for the detection and quantification of mineralization, which is a marker of osteoblast differentiation. However, this method presents a number of drawbacks, including the need to fix cells, which prevents additional measurements. Years ago, Calcein Green was proposed as an alternative to Alizarin Red, with the advantage to be directly detectable in live cells. However, the protocol was still time-consuming, and it never managed to replace Alizarin Red. Now, with more efficient imaging systems, we present a protocol using Calcein Green which provides significant advantages.
Results: The osteoblast mineralization was efficiently detected and accurately quantified in real time at any desired time point across the entire differentiation period, with a minimum time expenditure.
Conclusions: The combination of Calcein Green and the real-time imaging station IncuCyte ZOOM can efficiently replace the Alizarin Red method, and allows very accurate and time-saving assessment of the level and the dynamics of matrix mineralization.
Keywords: Alizarin red; Bone; Calcein green; Imaging; Mesenchymal; Mineralization; Osteoblast differentiation; Time-lapse photography.