Imagexpress micro confocal

H9c2 cells were seeded in 96-well plates with 2000 cells or 3000 cells per well. After certain treatments, cells were fixed with 4% paraformaldehyde solution (Sangon, Shanghai, China) for 20 min, permeabilized with 0.2% Triton X-100 for 10 min, blocked with PBST (PBS + 0.1% Tween-20) containing 1% BSA and 22.52 mg/mL glycine for 30 min, then incubated with primary antibody at 4 °C overnight. Subsequently, cells were incubated with fluorophore-coupled secondary antibody at room temperature for 2 h, and finally with 1 μg/mL of Hoechst 33,342 (Invitrogen, Carlsbad, USA) for 10 min to stain the nuclei. Images were acquired by the ImageXpress Micro Confocal (Molecular Devices, San Jose, USA) high-content imaging system using a 40× objective. Images were analyzed by MetaXpress (Molecular Devices) software using the Cell scoring module to obtain the average γ-H2AX fluorescent intensity and using the Granularity module to obtain the γ-H2AX or 53BP1 foci per cell. The primary antibodies involved were anti-γ-H2AX (Ser139) (1:500, CST (Danvers, USA), #9718) and anti-53BP1 (1:250, Abcam (Waltham, USA), #ab175933).

BMDMs were seeded in μ-Plate 96 Well Black (Ibidi) at a concentration of 1.25 x 10⁶ cells/ml. Cells were infected or stimulated for 8 and 24 hours and fixed with 4% PFA (Fluka). Cells were subsequently stained with 4′,6-diamidino-2-phenylindole (DAPI) (Molecular Probes) and Alexa Fluor 488 phalloidin (Molecular probes) to stain the nuclei and cytoplasm, respectively. Images were acquired using ImageXpress Micro Confocal (Molecular Devices) with a 40x objective. Parasite and cell number per well were quantified using MetaExpress custom Module Editor (Molecular Devices) as described previously (42 (link)).

AML12 cells stably expressing mitochondrial targeted ratiometric calcium sensor, 4mitD3-CPV^{69 (link)} or primary hepatocyte were plated in a black, clear bottom 96 well plate (3603; Costar), and transduced with 4mitD3-CPV adenovirus for 24 h. The cells are then washed, and replaced the medium with Ca²⁺-free Krebs-Ringer bicarbonate (KRB) buffer. Fluorescence signals were monitored using two emission filters, YFP (540 nm) and CFP (490 nm) by ImageXpress Micro confocal (Molecular Devices). Fluorescence images was recorded every 10 s, and 90 s later, 100 µM (final concentration) ATP was injected. YFP and CFP fluorescence ratio were quantified using MetaXpress software (Molecular devices). 4mitD3-CPV plasmid used for stable cell line preparation, and adenoviral construct was kindly provided by Dr. Kyu-Sang Park (Yonsei University Wonju College of Medicine, Wonju, South Korea). Cytosolic Ca²⁺ levels were measured using the Fura-2 QBT™ Calcium Kit (#R6139; Molecular Devices) as described in the manufacturer’s protocol. Briefly, cells were loaded with Fura-2 for 1 h and excited at 340 nm and 380 nm, with emission at 510 nm using FlexStation3 (Molecular Devices). Fluorescence signals were measured every 10 s and 60 s later, 100 µM ATP was injected.

The WBC (OD₆₀₀ ~ 0.3) was diluted with the same volume of broth medium containing BBR to give an approximate starting inoculum of 10⁸ CFU/ml and to obtain final BBR concentrations of 1/4 × MIC, 3/8 × MIC, 1/2 × MIC, or MIC. The control culture was added with the same volume of broth medium without BBR. The cultures were incubated further for 8 h, and cell growth was monitored spectrophotometrically (OD_600nm at 1 h intervals). Time kill tests of the above BBR concentrations were assessed by counting CFU (Lobritz et al., 2015 (link)). WBC was incubated in different concentrations of BBR at 37°C for 30 min. Bacterial cells were collected by centrifugation and washed two times with 0.85% sterile saline and then stained for 15 min using Live/Dead BacLight bacterial viability kit (Invitrogen, Eugene, OR, USA) according to the manufacturer's instructions and imaged by Image Xpress Micro Confocal (Molecular Devices LLC, Sunnyvale, CA).

Cells were fixed in 4% paraformaldehyde at room temperature for 0.5 h and permeated by 2% Triton X-100 for 10 min. After 30 min of blocking with PBST (1%BSA and 22.52 mg/mL glycine), FXR primary antibody (1:100) (Proteintech, 25055-1-AP) was added to incubate overnight at 4 °C, followed by adding fluorescent secondary antibody TRITC-conjugated Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00007-2) for 1 h. Then, Hoechst (1:1000) was added and incubated for 10 min to stain the cellular nucleus. Images were taken and analyzed by ImageXpress Micro Confocal (Molecular Devices, San Jose, CA, USA).