Deflex flow cytometer

Lung tissues from lung metastasis models were dissected, washed and digested with 2 mg ml^-1 collagenase type I (Worthington Biochemical), 2 mg ml^-1 collagenase type IV (Worthington Biochemical), and 0.2 μg ml^-1 DNase I (Sigma-Aldrich) in RPMI-1640 medium at 37 °C for 1 h on a shaker. The digestion process was terminated with RPMI-1640 medium containing 10% FBS. Then, the cell suspension was filtered through a 70 μm cell strainer, and RBCs were lysed.
For flow cytometry, a single-cell suspension was incubated first with FcR blocking antibody for 30 min at 4 °C and then with fluorophore-conjugated primary antibodies for 30 min at 4 °C for surface staining. Dead cells were excluded by staining with Fixable Viability Dye eFluor^TM (Thermo Fisher Scientific). Samples were injected into a Deflex flow cytometer (Beckman Coulter, Brea, CA, USA), and the data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA).

The change in cell cycle distribution was assessed using a cell cycle assay kit (KeyGen BioTECH, Jiangsu, China). First, PC cells were incubated with 0.2 μM nocodazole for 24 hours to synchronize PC cells in the G2/M phase. Thereafter, the culture medium was removed and synchronized cells were treated with different concentrations (0, 20, 40, and 80 μM) of ST for 48 hours. Further, PC cells were collected, immobilized in 70% ethanol overnight at -20℃, washed with PBS two times, stained with propidium iodide reagent, and detected using a DeFLEX flow cytometer (Beckman, USA). The results were analyzed using Flwo JO (version 7.6.1).