Calcein am stock solution

Calcein-AM (calcein acetoxymethyl ester) was used to stain the adherent viable cells on the surface of nanocomposites after 1 and 5 days of incubation. 5 μl of calcein-AM stock solution (40 mM concentration, Sigma Aldrich, St. Louis, MO, USA) was added to 10 ml DBPS and then added to the crosslinked nanocomposite disks (100,000 cells/specimen or 3 x 10⁵ cells/cm²). The disks were incubated at 37°C. After incubation for 30 minutes in dark, samples were washed with DPBS prior to confocal imaging. Fluorescence images were recorded under an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Wells of a 96-well pate (TCPS control) containing initially 100,000 (3.12 x 10⁵ cells/cm²) seeded cells served as a positive control whereas blank wells containing media served as a negative control. Sample size was n=3 for calcein-AM staining.

In order to stain viable cells for fluorescence microscopy, calcein-acetoxymethyl ester (calcein-AM) staining that has been widely used to selectively stain metabolically active living cells was performed [6 (link), 43 ]. 5 μL of calcein-AM stock solution (40mM, Sigma Aldrich, St. Louis, MO, USA) was mixed with 10 mL DBPS to prepare a working concentration of 4 μM. 1 mL of calcein-AM working solution. Then, the working solution was added to each well containing nanocomposite discs after MC3T3 cell culture for 24-h) and incubated at 37°C for 25 minutes. Samples were rinsed with DPBS and placed in 35-mm glass bottom petri dishes (Mattek Corp., Ashland, MA, USA) for confocal fluorescence microscopy at 485 excitation wavelength and 530 nm emission wavelengths. TCPS wells with same surface area (compared to nanocomposite samples), after seeding at 5000 cells/well and 24-h incubation, served as positive (live) controls. PPF discs after seeding at cell density of 5000 cells/well and 24-h incubation were treated with lysis solution and served as negative controls. Two samples (n=2) were stained for each experimental group.