The mitochondria, actin filaments and the nucleus of treated/untreated pre-osteoblasts and pre-osteoclasts were stained as described previously [20 (link)]. Briefly, mitochondria were stained using MitoRed dye, the F-actin filaments with Phalloidin-Atto 488 and the cells nuclei with 4′,6-diamidino-2-phenylindole DAPI (all from Life Technologies, Carslbad, CA, USA).
Briefly, the cells after incubation with the PMMA and its combination, were incubated for 30 min with MitoRed solution at concentration 1:1000 at 37 °C and fixed with 4% PFA (POCh, Gliwice, Poland). Then, the cells were stained with Phalloidin-Atto 488 for 45 min at RT and then stained with DAPI. Visualization was made by a confocal microscope (Leica TCS SPE, Leica Microsystems, Wetzlar, Germany) at 0.5 µm steps up to a final depth of 25 µm. Images were captured at magnification 630× and analyzed using Fiji New ImageJ with Colour Pixel Counter plugin version 1.52 developed by Wayne Rasband from NIH, USA. Each photograph was taken at least three times independently.
Briefly, the cells after incubation with the PMMA and its combination, were incubated for 30 min with MitoRed solution at concentration 1:1000 at 37 °C and fixed with 4% PFA (POCh, Gliwice, Poland). Then, the cells were stained with Phalloidin-Atto 488 for 45 min at RT and then stained with DAPI. Visualization was made by a confocal microscope (Leica TCS SPE, Leica Microsystems, Wetzlar, Germany) at 0.5 µm steps up to a final depth of 25 µm. Images were captured at magnification 630× and analyzed using Fiji New ImageJ with Colour Pixel Counter plugin version 1.52 developed by Wayne Rasband from NIH, USA. Each photograph was taken at least three times independently.