Lsr 2 flow cytometry machine

CLBL‐1 and MDCK cells were treated with AZD1480 and CYT387 as described in the cell viability assay. JAK inhibitor‐treated CLBL‐1 and MDCK cells were stained with Annexin V and SYTOX Red dead cell staining and were evaluated by flow cytometry. After 72 hours of drug treatment, cells treated with DMSO, AZD1480 (2 μm and 5 μm), or CYT387 (1 μm and 5 μm) were collected and washed once with Dulbecco's phosphate‐buffered saline (DPBS) and once with 1 × Annexin V binding buffer (0.1 m Hepes/NaOH, 1.4 m NaCl, and 5 mm CaCl2). Cells at a concentration of 1 × 10⁶ cells/mL were resuspended in the binding buffer solution and stained with fluorescein isothiocyanate (FITC)‐conjugated Annexin V (eBioscience19; 1 : 40) and SYTOX DNA binding dye (Thermo Fisher Scientific10; 1 : 1000). Samples were analyzed with BD LSRII flow cytometry machine (BD Biosciences17), and results were analyzed by FlowJo v10.0.7 software18.

Tumor cells were cultured in 5% FBS in DMEM following standard cell culture protocol. Hoechst 33342 (ThermoFisher Scientific; #H1399) was added directly to the cell culture medium with the final concentration of 10 μg/mL 1 hour before sample collection. Cells were incubated with Hoechst 33342 at 37°C for 1 hour. Then, cells were prepared to single cell suspension by using trypsin and washed with PBS twice. Cells were resuspended in flow buffer (1% FBS in PBS) for flow analysis in a BD LSR II flow cytometry machine. Flow results were analyzed by using the FlowJo software to assess the percentage of cells in G1 phase and S-G2-M phase.