Figure 2
Confocal Images showing dual staining and Raman spectra of MOVAS-1 monolayers. Monolayers of MOVAS-1 cells were grown to confluence (day 0) and then switched to calcification media (2.4 mM Ca and 1.4 mM Pi) for 7 days, changing media on alternate days. The cell monolayers were treated sequentially with Fluorescein-BP (1 μM, 2 hours; green); CellMask Orange (500 nM, 10 minutes, pink); NBF (10%, 15 minutes); Triton X (0.05%, 3 × 5 minutes); DAPI (300 nM, 5 minutes, blue); then imaged on an Zeiss Confocal LSM710 microscope (λex = 488 nm, 554 nm and 350 nm). Images are representative of at least 3 repeats. Scale bars = 100 µm. (A) Incubation with Fluorescein-BP and DAPI. (B,C) Incubation with Fluorescein-BP, CellMask Orange and DAPI. (D) Monolayers of MOVAS-1 cells were grown to confluence (day 0) on CaF2 slides and then switched to either control media or calcification media (2.4 mM Ca and 1.4 mM Pi) for 7 days, changing media on alternate days. The cell monolayers were fixed with NBF (10%, 15 minutes); and imaged using an InVia Renishaw Raman instrument (785 nm, 30 s, 50%). Following imaging, the cell monolayers were incubated with Alizarin S (2%, 10 minutes). Red dashed line indicates peak at 960 cm−1 for HAP while the black dashed line indicates the inherent cellular peak at 1003 cm−1 for phenylalanine. Images shown are representative of at least 3 independent experiments yielding comparable results.
