Phalloidin atto 488

Cellular morphology was visualized at week1 and week2 using fluorescence microscopy. Briefly, cells and cell-laden constructs were fixed with 4% paraformaldehyde (PFA) in PBS (pH7.4) for 10 min at room temperature (RT). After rinsing with PBS for three times, the samples were placed in a permeabilization solution with 0.1% (v/v) Triton X-100 for 10 min and rinsed again with fresh PBS for three times. The cells and constructs were then blocked with 1% BSA in PBS for 1 hour at RT. Immunostaining with primary antibody rabbit monoclonal FSP1 (Abcam, Cambridge, UK) (1:400) and Alexa Fluor 647-conjugated donkey anti-rabbit IgG (Life technologies, Carlsbad, CA) (1:1000) was performed at 4 °C with gentle shaking for overnight, then counterstained with Phalloidin-Atto 488 and Hoechst 33258 (Life technologies, Carlsbad, CA) to visualize the f-actin and nuclei, respectively. The cells and cell-laden constructs were visualized using a Zeiss LSM 700 laser confocal microscope (Carl Zeiss Micro-Imaging GmbH, Germany).

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.