Nucblue live readyprobes reagent hoechst 33342

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).

5 × 10⁴ Flp-In™ T-Rex™ cells expressing SNAP-editases were seeded on glass cover slips coated with poly-(d)-lysine hydrobromide (Sigma-Aldrich, 0.1 mg/ml in H₂O) in DMEM, FBS (10%), B, H with or without doxycycline (10 ng/ml final conc.). Cells were incubated with 200 μl of DMEM, FBS (10%) containing 2 μl NucBlue™ Live ReadyProbes™ Reagent Hoechst 33342 (ThermoFisher Scientific) and O-acetylated benzylguanine fluorescein isothiocyanate (ac-BG-FITC, 2 μM final conc.). After fixation with formaldehyde (3.7% final conc.) and permeabilization with Triton X-100 (Carl Roth, #3051.3, 0.1% final conc.), cover slips were mounted on microscope slides using Dako Fluorescence Mounting Medium (AgilentDako) and dried over-night at 4°C. Images were taken with an AXIO Observer.Z1 (Zeiss), Colibri.2 light source under 63x magnification.