Novoexpress flow cytometer

Single cells of SVF fraction were resuspended with PBS and stained with zombie NIR™ dye (#423105, Biolegend, USA), blocked with FcX Blocking CD16/32 (#156604), and dyed with a mix of surface marker antibodies. Cells were washed with cell staining buffer (#554657, BD Biosciences, USA), suspended, and transferred to a flow tube. Data was collected on a NovoExpress flow cytometer (Agilent, USA) and analyzed using Flowjo_V10 software (Tree Star).

Cell cycle analysis was carried out using the Cell Cycle Detection Kit (Invitrogen, USA) following the manufacturer’s instructions after the cells had been co-cultivated with mesothelial cells in a transwell plate, cultured with the conditioned media, or treated with DMSO or MRK-003. Trypsinization was used to collect OvCa cells, which were then washed in ice-cold PBS and fixed in a 75% ice-cold ethanol solution. Before labeling, cells were gently resuspended in ice-cold PBS. Next, propidium iodide (PI) was incubated with the cells for 30 minutes at room temperature. Following that, data were examined by NovoExpress software and the Novo Express flow cytometer (Agilent, USA).