Semi-quantitative fluorescence analysis of calcein binding as a measurement of in vitro mineralization

We describe a new, highly sensitive semiquantitative method for rapid measu rement of in vitro mineralization using calcein. Fluorescence analysis of t he calcein bound to the calcium phosphate (hydroxyapatite) allows direct qu antitation of extracellular matrix mineral content in monolayer cultures of bone-forming cells such as primary osteoblasts or osteosarcoma cells. Oste osarcoma cell lines UMR 106 and SaOS-2 were used to demonstrate that qualit atively, calcein was bound to the same regions of the mineralized cell mono layer as seen by conventional histological staining with von Kossa or Aliza rin Red S. Moreover: total bound calcein could be quantitated by direct flu orescence analysis using a Cytofluor II plate reader. Changes in cell monol ayer calcein fluorescence were shown to correlate well with direct colorime tric measurement of acid-solubilized Ca+2 from parallel cultures. Relative mineral quantitation by calcein fluorescence is rapid and more sensitive th an colorimetric Ca+2 assays, can be performed directly on unfixed or fixed cell monolayers, and does not require the use of radioisotopes. The cell mo nolayer remains intact and potentially available for further analysis.