Abstract
BackgroundIn 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.ResultsThe 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.ConclusionsThe 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.
Highlights
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
Primary mesenchymal stem cells induced to differentiate to osteoblasts were incubated at 37 degrees and monitored in the presence of Calcein Green for 14 days in the IncuCyte ZOOM
At the end of the differentiation period the cells previously treated with Calcein Green were fixed and further stained with Alizarin Red
Summary
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. This method presents a number of drawbacks, including the need to fix cells, which prevents additional measurements. The protocol was still time-consuming, and it never managed to replace Alizarin Red. with more efficient imaging systems, we present a protocol using Calcein Green which provides significant advantages. The final stage, matrix deposition and mineralization, is traditionally used as readout of the differentiation efficiency, but its quantification has been cumbersome and requires termination of the cultures. The obtained absorbance has to be normalized to the cell number, most commonly by measurement of total protein in a parallel sample, which increases variability, and the method requires several hours of lab work
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