Ge in cell analyzer 2200

Uterine epithelial cells were seeded at a concentration of 5 × 10⁴ cells/well on a 24-well plate and then treated as mentioned above. Next, the cells were washed in PBS and fixed in 2% paraformaldehyde for 20 min at RT. The cells were then washed in PBS, incubated with rhodamine phalloidin (#R415, Thermo Fisher) for 20 min in the dark at RT, and then washed in PBS. The intensity of fluorescence was detected using high-content imaging with a GE IN Cell Analyzer 2200 (GE Healthcare Life Sciences, Chicago, IL, USA) and quantified with IN Carta^TM image analysis software (GE Healthcare Life Sciences).

Cells were plated at 10,000 cells per well in black 96-well microplates with glass bottom (Corning) in normoxic and hypoxic media conditions. Prior to imaging, cells were treated with 10nM tetramethyl rhodamine (TMRM) for 30 min, protected from light in a 37°C/5% CO2 incubator. Fluorescent images of mitochondrial membrane potential were obtained at 542.0 nm (27.0 nm bandpass) excitation and 587nm (45nm bandpass) emission on a GE INCell Analyzer 2200 (GE Healthcare). Sequential qualitative images were taken in 10 fields of view for each channel in each well at 37°C. Images were analyzed using GE’s InCarta software version 1.6. Multiple parameters were collected from each plate by creating custom “masks” that captured the TMRM fluorescence signal, allowing for subsequent quantification. Data are expressed as intensity (the mean pixel value under the mask)—background (mean pixel value for the local background) in Arbitrary Units (A.U.) ± SEM.