Cell cycle analysis kit

Procedures of cell cycle assay were shown as follow: cells from different groups were grown in 6-well plates and kept for 48 h. Then, we had them washed with ice-cold PBS and fixed them in 70% (v/v) ice-cold ethanol solution overnight at 4°C; these cells were analyzed in the following day with flow cytometry following the instruction of cell cycle analysis kit (Sigma, MO, USA). Information of cell cycle was determined with ModFit LT 4.0 software.

In order to investigate the changes in the cell content, cell cycle analysis was accomplished by flow cytometry method in cells treated with BiFe₂O₄@Ag nanoparticles. According to procedure of Sigma-Aldrich (USA) Cell Cycle Analysis Kit, the AGS cells were cultured with a density of 5 × 10⁵ in a 6-well plate and subjected to 67 μg/mL of BiFe₂O₄@Ag nanoparticles for 24 h in a CO₂ incubator. In following step, the cells were collected and washed with PBS buffer. Afterward, cold ethanol (70%) was used for fixation, and propidium iodide was used to stain of the cells. Further, the cells were treated with RNase A (100 µg/mL). In the final step, a flow cytometer (ParTec™, Germany) was used to measure the DNA content of the cells.

The cell proliferation ability was examined using Cell Counting Kit-8, the cell migration ability was assessed using a wound scrape assay, and the cell invasive ability was examined by using matrigel-coated invasion chambers [29 (link), 30 (link)]. For the cell cycle assay, the cells from different groups were inoculated in 6-well plates and cultured for 48 h, then were washed with ice-cold PBS and fixed in 70% (v/v) ice-cold ethanol solution overnight at 4 °C, then in the following day, these cells were analyzed by using flow cytometry according to the instruction of cell cycle analysis kit (Sigma, MO, USA), the cell cycle information was analyzed using ModFit LT 4.0 software.

Cell proliferation assays were performed using a Cell Counting Kit-8 (CCK-8, APExBio, USA). Proliferation rates were determined 0, 24, 48, 72, and 96 h after infection, and the absorbance was measured at 450 nm following the manufacturer-recommended protocol. The apoptosis rate was confirmed by flow cytometry (FCM) using an Annexin V-FITC/PI apoptosis detection kit (Elabscience Biotechnology Co., Ltd., Wuhan, China). Cell cycle analysis was performed using a Cell Cycle Analysis Kit (Sigma, St. Louis, MO, USA).