Operetta cls high content screening system

Cell viability was assessed using Calcein-AM/PI staining assays (Beyotime, Shanghai, C2015M). DAPI (Invitrogen, Carlsbad, CA) was used to stain the cell nuclei. A total of 1×10⁶ Hep3B or Huh7 cells were plated into 96-well plates for 24 h to allow for cell attachment before being incubated for an additional 48 h with various concentrations of the tested compounds. The concentrations of gambogenic acid or DMSO (solvent of gambogenic acid) were 0 μM (Ctrl), 0.1, 2, 10, 50, and 100 μM. The concentrations of alisertib or DMSO (solvent of alisertib) were 0 μM (Ctrl), 0.1, 1, 10, 50, and 100 μM. For slope analysis, a total of 1×10⁶ Hep3B or Huh7 cells were plated into 96-well plates for 24 h to allow for cell attachment before being incubated for additional time (0, 12, 24, 36, 48, and 60 h) with gambogenic acid (2 μM) and alisertib (10 μM), respectively. The cells were then cultured with Calcein-AM, PI and DAPI at 37°C for 30 min according to the manufacturer’s protocol. Subsequently, images were captured using a PerkinElmer Operetta CLS High Content Screening System. All assays were conducted at least three times.

Cell viability was assessed using Calcein-AM/PI staining assays (Beyotime, Shanghai, C2015M). DAPI (Invitrogen, Carlsbad, CA) was used to stain the cell nuclei. A total of 1×10⁶ Hep3B or Huh7 cells were plated into 96-well plates for 24 h to allow for cell attachment before being incubated for an additional 48 h with various concentrations of the tested compounds. The concentrations of gambogenic acid or DMSO (solvent of gambogenic acid) were 0 μM (Ctrl), 0.1, 2, 10, 50, and 100 μM. The concentrations of alisertib or DMSO (solvent of alisertib) were 0 μM (Ctrl), 0.1, 1, 10, 50, and 100 μM. For slope analysis, a total of 1×10⁶ Hep3B or Huh7 cells were plated into 96-well plates for 24 h to allow for cell attachment before being incubated for additional time (0, 12, 24, 36, 48, and 60 h) with gambogenic acid (2 μM) and alisertib (10 μM), respectively. The cells were then cultured with Calcein-AM, PI and DAPI at 37°C for 30 min according to the manufacturer’s protocol. Subsequently, images were captured using a PerkinElmer Operetta CLS High Content Screening System. All assays were conducted at least three times.

The assay was performed using the Seahorse XF Cell Mito Stress Test kit (Agilent). Cells were differentiated as described above (14 days monoculture microglia like) and, in a Seahorse XF96 cell culture microplate, 50,000 cells (480,000 cells/cm²) were seeded two days prior to the experiment. On the day of the experiment, the medium was replaced with 180 µl media for the mitochondrial respiration stress test (base medium + 2 mM L-glutamine (Thermo Fisher Scientific), 1 mM sodium pyruvate (Thermo Fisher Scientific), and 0.45% glucose (Sigma Aldrich), pH 7.4). Prior to the experiment, the cell culture microplate was incubated for 1 h at 37 ˚C in a non-CO₂ incubator. The assay was performed using a Seahorse XFe 96 Analyzer and compounds (prepared according to the assay manual) were injected sequentially (1 µM oligomycin, 2 µM FCCP, and 500 mM rotenone/antimycin A) and the oxygen consumption rate (OCR) measured three times before treatment and after every injection. Data was processed and analyzed using the Seahorse Wave software. For normalization, cells were fixed with 4% PFA at 37°C for 15 min and nuclei stained with Hoechst 33342 for 15 min, before washing twice with PBS. The Operetta CLS high-content screening system (Perkin Elmer) was used for imaging nuclei counted and the ratio between cell types calculated.