Cell f software

To measure intracellular ROS production, the cells growing on coverslips were loaded with 5 μM dihydrofluorescein diacetate (H2DCF-AC; 20 min, 37°C), thoroughly rinsed with Hank’s Balanced Salt Solution, and mounted on slides. Live cells were examined with the Olympus IX71 fluorescence microscope equipped with the Cell^F software (Olympus). The fluorescence of the dye was excited at 488 nm for 500 ms. the corrected total cell fluorescence (CTCF) of individual cells was calculated using the Cell^F software (Olympus), and presented in percentage frequency (“cumulative distribution”) graph. The experiments were repeated three times with similar results and representative data from one experiment is shown in Figure 4. Collected data was analyzed as described above (“Immunocytochemistry”).

The growth curve of stem cells on NPs and on CTR was measured at 3, 7, 14, and 21 Days (D). To count the cell nuclei, at each time point, cultures were harvested and fixed in 4% paraformaldehyde and rinsed with PBS, and coverslips were mounted and nuclei were counterstained with Vectashield Antifade Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories Inc., Burlingame, CA, USA). The quantification of the number of cells on NPs and CTR was carried out considering 10 different photos (10× magnification) to cover the total NP deposition area, and the DAPI-stained nuclei were counted by using the Cell^f software (Soft Imaging System, Olympus, Münster, Germany, version 2.5, Accessed in 2006). Images were acquired using a fluorescence microscope (Eclipse-TE2000-S, Nikon, Tokyo, Japan) equipped with the F-ViewII FireWire camera and Cell^f software (Soft Imaging System, Olympus, Münster, Germany, version 2.5, Accessed in 2006). The stem cell proliferation is expressed as a mean of 3 independent experiments ± SEM.

To measure intracellular ROS production the cells growing on coverslips were loaded with 5 μM dihydrofluorescein diacetate (H2DCF-AC, Sigma; 20 min, 37 °C), thoroughly rinsed with Hank’s Balanced Salt Solution, and mounted on slides. Live cells were examined with the Olympus IX71 fluorescence microscope equipped with the Cell^F software (Olympus). The fluorescence of the dye was excited at 488 nm for 500 ms^{58 (link)}.
The Ca²⁺-sensitive fluorescent dye Fluo-3-AM was used to detect the relative change of the Ca²⁺-dependent fluorescence in cells. Cells were loaded for 30 min with 5 µM dye in amino-acids- and serum-free medium, washed twice and incubated for 15 min in full culture medium (de-esterification of the dye). The fluorescence was measured with the FV-1000 confocal microscope (Olympus) using excitation and emission wavelengths of 488 and 525 nm, respectively. The measurements were taken from at least 70 cells (usually over 100 cells) from randomly selected areas. All cells observed in the areas were used for measurements. The corrected total cell fluorescence (CTCF) of individual cells was calculated using the Cell^F software (Olympus) and presented in percentage frequency graphs (“cumulative distribution”) graphs. The experiments were performed in triplicate, with similar results.