Cellquest flow cytometry software

DCFH‐DA, a cell‐permeable fluorescent probe, was used to determine the intracellular ROS levels of the LoVo cells. The cells were cultured at a density of 10⁵/well on 24‐well plates for 24 h to allow adhesion. After that, cell media were substituted with serial concentrations of LRPS containing LRAC (20 µg/ml), or H₂O₂ designated as positive control. The cells were then incubated for 24 h, followed by PBS washing of two times, and incubated with DCFH‐DA probe (10 µM) at 37°C for 20 min. The fluorescence intensity generated by DCFH‐DA in the LoVo cells was recorded by the flow cytometry (excitation: 485 nm, emission: 525 nm). The result was analyzed and visualized using CellQuest‐Flow Cytometry Software (Becton Dickinson).

Cells for apoptosis analysis were treated according to the procedure as described in cell cycle analysis section. An apoptosis detection kit (BestBio) was employed to analyze apoptotic distribution of the cells. PS (phosphatidylserine) usually found on the inside of the cell membrane and nuclear DNA were used as the biomarkers for the assay. Simultaneous quantification of vital and apoptotic cells was achieved by using dual dying (Annexin V‐FITC (fluorescein isothiocyanate) and PI, targeting PS and nuclear DNA, respectively). Briefly, cells of 10⁶ were collected and washed twice with PBS, followed by being resuspended in 400 µl of binding buffer with the addition of Annexin V‐FITC (5 µl) and PI (10 µl). The resulting mixtures were kept in dark at 4°C for 10 min, and the cells were loaded on the flow cytometer for analysis. The result was analyzed and visualized using CellQuest‐Flow Cytometry Software (Becton Dickinson).