Ibitreat 1.5 polymer slides

HCT116 and HCTR cells were seeded at a density of 0.5 × 10⁵ cells/mL in 200 µL medium in 8-well ibiTreat 1.5 polymer µ-slides (80826, ibidi GmbH, Gräfelfing, Germany) and allowed to adhere overnight. The cells were exposed to DMSO, BOLD-100, 2-DG, or their combination in the indicated concentrations for 24 h. The cells were fixed with 4% paraformaldehyde (PFA)/PBS (pH 7.4) for 30 min. and permeabilized in PBS/0.5% v/v Triton X-100 (pH 7.4) for 20 min at room temperature. The cells were blocked in PBS (pH 7.4) containing 20% w/v BSA for 20 min and stained with primary anti-LC3B rabbit monoclonal antibody (1:400, #12741 from Cell Signaling Technology, Danvers, MA, USA) in PBS (pH 7.4) containing 2% w/v BSA at 4 °C overnight. The cells were washed in PBS (pH 7.4), incubated with Alexa Fluor 488-labeled goat anti-rabbit IgG, (1:500; Invitrogen, Thermo Fisher, USA) for 1 h at room temperature in the dark, and nuclei were counterstained with Hoechst 33258 (blue) (1 mg/mL in PBS pH 7.4, Sigma-Aldrich). After a final washing step, high-resolution live-cell spinning-disc images were taken, as described above.

HCT116 and HCTR cells were seeded at a density of 0.5 × 10⁵ cells/mL in 200 µL medium in 8-well ibiTreat 1.5 polymer µ-slides (80826, ibidi GmbH, Gräfelfing, Germany) and allowed to adhere overnight. The cells were exposed to DMSO, BOLD-100, 2-DG, or their combination in the indicated concentrations for 24 or 72 h. After the drug incubation period, the cells were stained with 0.5 µM Lysotracker Red (L7528, Life Technologies, CA, USA) and Hoechst 33258 (blue) (1 mg/mL in PBS pH 7.4, Sigma-Aldrich). High-resolution live-cell spinning-disc images were taken using an IX83 P2ZF microscope (Olympus Austria GmbH, Vienna, Austria) with a UPLXAPO O 60 oil-immersion objective at 192× magnification. Image acquisition was performed using the OLMYPUS cellSens Dimension 3.1 imaging software (OLYMPUS CORPORATION, Tokio, Japan).