Phalloidin rhodamine solution

In order to observe the podosome belt of RANKL‐induced osteoclasts, BMMs were cultured on fetal bovine serum (FBS)‐coated cover glasses in a 96‐well plate, and cells were treated with Rob (0, 1, or 2 μM) as described above. Cells were fixed in 4% PFA for 8 min, infiltrated with 0.1% (v/v) Triton X‐100 for 10 min, blocked with 3% bovine serum albumin (BSA) for 1 h, incubated 12 h with anti‐vinculin (Sigma‐Aldrich, St. Louis, MO, USA), washed in PBS, and finally cultured with Alexa Fluor 488 (Invitrogen, Waltham, MA, USA). F‐Actin ring was stained with Rhodamine Phalloidin solution (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h. The cells were washed with PBS and subsequently stained with 4,6‐diamidino‐2‐phenylindole (DAPI, Santa Cruz Biotechnology, Dallas, TX, USA), followed by visualization with a confocal microscope (Nikon, Tokyo, Japan).

To evaluate the organization of F-actin in the gastric adenocarcinoma cells during exposure to heat-treated H. pylori a fluorescent staining was performed, at condition corresponding to the Young’s modulus measurements. To do so, 1 × 10⁴ of the cells were seeded onto glass coverslips and incubated overnight at 37 °C in an atmosphere containing 5% CO₂. The next day, cells were washed with PBS and incubated with 1 × 10⁵ CFU/mL of heat-inactivated bacteria suspended in serum and antibiotic free growth medium for 0.25–72 h. Then, cells were washed twice with PBS and fixed in 3.7% paraformaldehyde for 15 min, RT. Cells were washed again and permeabilized with 0.1% Triton X-100 for 10 min, RT. To block unspecific binding, cells were incubated for 30 min at RT with 1% bovine serum albumin (BSA) in PBS. After another wash, cells were incubated with rhodamine phalloidin solution (Thermo Fisher Scientific, Rockford, MI, USA) for 1 h, RT, in the dark. In the next step, cells were counterstained with NucBlue™ Live ReadyProbes™ Reagent Hoechst 33342 (Thermo Fisher Scientific) for 20 min, RT, protected from light. Preparation was mounted with anti-fade fluorescent mounting solution (Abcam, Cambridge, UK) and covered with glass coverslip. Cell structure visualization was performed using Leica DM4 B fluorescent microscope (Wetzlar, Germany).

After 96 h of culture, some samples were immersed in Rembaum solution [66 (link)] for 1 h then rinsed three times with demineralized water. They were observed using SEM (Philips XL30 ESEM-FEG, Eindhoven, The Netherlands; quanta FEG 250, FEI, Hillsboro, OR, USA; Hitachi S-3400N, Tokyo, Japan) after gold coating.
Others were stained to observe the alignment of the actin filaments using fluorescence microscopy. Briefly, samples were fixed in paraformaldehyde (Agar Scientific, Stansted, UK), immersed for 45 min in a rhodamine/phalloidin solution (5 units/mL, Invitrogen, Waltham, MA, USA) then rinsed with PBS. The nuclei of the cells were also stained with DAPI (1 g/L, Invitrogen, Waltham, MA, USA). Samples were then observed (Leica Microsystems, Wetzlar, Germany) with excitation and emission wavelengths of 540/565 nm (rhodamine/phalloidin) and 358/461 nm (DAPI).