Evaluating GSSG Liposomes' Impact on Cancer Cell Detachment

The effect of GSSG liposomes on cancer cell detachment was investigated by a controlled trypsinization procedure with minor modification^{19 (link)} for NCI-H226 and B16-F10 cells. Cells (100 000 cells/well) plated in RPMI medium with 10% FBS and 1% penicillin/streptomycin in a 12-well plate were allowed to attach for 24 hours before being treated with GSSG liposomes, blank liposomes, or aqueous GSSG for 24 hours in a CO₂ incubator at 37°C. After treatment, cells were washed with DPBS without calcium and magnesium salts (1 mL 3×). Dulbecco’s Phosphate-Buffered Saline was removed completely before addition of a diluted trypsin solution (0.005% trypsin/5 mM EDTA, 0.5 mL). The plates were shaken slowly. At the end of the experiment, 2 mL of FBS-containing medium was added to stop the proteolytic action of trypsin. The supernatant was transferred to a conical tube, and cells that remained attached were photographed under a Fisher Micromaster Microscope (Waltham, MA). The supernatant was centrifuged and resuspended in 0.1 mL of the RPMI medium before being counted by a Cellometer Auto T4 Plus Cell Counter (Nexcelom Bioscience, Lawrence, MA) for any cells present in the supernatant.