Figure 1.
The relativity cell viability, alkaline phosphatase, and carbonic anhydrase activities during cell culture.
The activity of each treatment at 5 days was normalized to 1.
Figure 2.
Micrographs of the mantle cells at 25 days.
(A) and (B) Cells labelled with fluo-3 AM (magnification 200x); (C) and (D) cells labelled with calcein (magnification 400x); (E) and (F) cells containing crystals (magnification 630x), as indicated with arrows. White light in (A), (C) and (E); fluorescent light in (B) and (D); polarized light in (F).
Figure 3.
The expression and secretion of shell matrix proteins by mantle cells.
(A), (B) and (C) The relative expression levels of ACCBP, nacrein and Pif80 during cell culture, respectively. (D), (E) and (F) Western blot analysis of secreted proteins using antibodies against ACCBP, nacrein and Pif80, respectively. Uncultured medium in lane 1 as a control, cell culture medium at 10, 15, 20 and 25 days in lanes 2, 3, 4 and 5, respectively. (G) and (H) The effects of uncultured medium and medium from cell culture at 25 days on calcium carbonate crystallization, respectively. The aggregation and morphology of calcites were affected by the medium from cell culture. (I) Raman spectra of the crystals formed in (G) and (H); the characteristic peaks of calcite are at 153, 282, 712 and 1086 cm−1. Scale bar 20 µm.
Figure 4.
Backscatter electron imaging combined with energy dispersive X-ray spectral analysis of the mantle cells.
In the Backscatter electron (BSE) mode, elements with a higher atomic number scatter more electrons (such as calcium and strontium), thus appearing lighter, whereas carbon appears black. (A), (B), (C) and (D) SEM-BSE images of cells at 10days, 15 days, 20 days and 25 days, respectively, in Pf-CM2.5. (E) and (F) SEM-BSE images of cells at 25 days in Pf-C10M50 and Pf-S10M50, respectively; the intracellular crystals are indicated with white arrows. (G) Energy dispersive X-ray spectral (EDS) of the cells in (A), the elemental composition of the cells consisted of C and O. The black arrow on the SEM image indicates the sample point of the EDS. (H) EDS of the crystals in (B), (C) and (D). The elemental composition of the crystals is C, O, Mg, P and Ca. (I) and (J) EDS of the crystals in (E) and (F), respectively. The elemental composition of the crystals is C, O, Mg, P and Ca in (E) and C, O, Mg, P, Ca and Sr in (F). The white arrows on the SEM image indicate the sample point of the EDS. Scale bars 10 µm.
Figure 5.
FTIR and XRD of intracellular crystals showing characteristic bands indicating the presence of ACC.
(A) FTIR spectra of powdered intracellular crystals; the broad bands at 580,1064,1235 and 1544 cm−1 are not related to a distinct calcium carbonate polymorph (calcite or aragonite). (B) X-ray powder diffraction (XRD) pattern of intracellular crystals (Cell) and synthetic ACC (ACC); the intracellular crystals and synthetic ACC exhibit the same characteristic bands for ACC.