Real-Time Vital Mineralization Detection and Quantification during In Vitro Osteoblast Differentiation

Abstract

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.

Fig. 1

Comparison of the efficiency of calcein versus Alizarin Red to detect mineralized areas of osteoblast culture. a mineralization pattern detected by Olympus IX2-UCB Fluorescent microscope, phase-contrast (left panel, calcium hydroxyapatite deposits appear like yellowish structures at phase-contrast); the same section on the plate visualized by calcein by fluorescent setting (middle panel); overlap between the phase-contrast and fluorescent images in composite setting (right panel), scale bars 2000 μm. Representative pictures are shown, n = 3. b Calcein staining and Alizarin Red staining of the same mineralized areas detected by IncuCyte ZOOM: note that the phase-contrast objective of the IncuCyte ZOOM does not show the red color of Alizarin Red, however the pattern of the staining is clearly visible in gray scale and can be compared to the calceinpattern; representative pictures are shown, n = 3

Fig. 2

Mineralization dynamics of differentiating osteoblasts treated with osteogenic cocktail (OGC), detected by Calcein. a Fluorescent images of the cell culture obtained by IncuCyte ZOOM at day 7, day 10 and day 14 of differentiation. Last row: vehicle (ethanol) treated cells, showing no differentiation, representative pictures are shown, n = 3. b Mineralization curve generated automatically by the IncuCyte ZOOM based on the green object count per image. The cells were scanned automatically every 3 h for two weeks, representative experiment is shown, n = 3, SE

Fig. 3

Comparison of quantification of mineralization obtained either by Alizarin Red (a) or calcein (b). MSCs were transfected by an siRNA against PPP2R2C and the mineralization was quantified on day 12 of osteoblast differentiation a Measurement of extracted Alizarin Red, b Measurement of calceinin cultures in the IncuCyte ZOOM. MSCs transfected with negative control siRNA were used as a control, n = 2, SE