Annexin 5 fitc pi

Cells were incubated with different concentrations of DCZ0415 for 24 h.
For cell cycle assay: cells were harvested, followed by washing twice with PBS, and were fixed overnight with 70% ethanol. Cell samples were stained by propidium iodide (PI), and the intensities were detected using flow cytometry (FACSVerse, BD, USA) by Analysis and Testing Center, Nanjing Medical University.
For apoptosis assay: supernatant DMEM medium and cells were collected, centrifuged 3 min, 800 g, and the pellets were washed twice by PBS. Cells were resuspended in 400 μL of precooled 1×binding buffer and stained by Annexin V-FITC/PI (Yeasen, Shanghai, China) for 30 min. Then cell suspension was detected on flow cytometry (FACSVerse, BD, USA) analysis and Testing Center, Nanjing Medical University.

At 48 h after coculture with M1 macrophages for 48 h, HK-2 cells were collected. The cells were fixed with 70% ethanol. The fixed cells were washed with PBS and then treated with DCFH-DA according to the Reactive Oxygen Species Assay Kit (Yeasen, Shanghai, China). In the apoptosis experiment, Annexin V-FITC/PI (Annexin V-FITC/PI Apoptosis Detection Kit, Yeasen, Shanghai, China) was used to label apoptotic cells. The stained cells were analyzed using a Becton Dickinson flow cytometer (Franklin Lakes, NJ).

To further explore the synergistic antitumor effects of 2-BP/CPT-PLNs in vitro, we studied their influences on the cell cycle and induced cell apoptosis. Briefly, B16-F10 cells were cultured in 12-well or 6-well plates and conducted by pipette tip to achieve a wound for further incubation with 2-BP, CPT, CPT + 2-BP, and 2-BP/CPT-PLNs for 24 h. The cell cycle arrest and apoptosis induced by 2-BP/CPT-PLNs were analyzed by FCM. The resulting cells were collected, fixed with 70% ethanol (v/v), washed with ice-cold PBS, and stained with PI solutions containing RNase A (Beyotime) for FCM measurements to analyze cell cycle ratios. Similarly, the resulting cells were harvested and immediately stained with Annexin V-FITC/PI (Yeasen) to analyze the cell apoptosis by FCM.
B16-F10 multicellular tumor spheroids (MCTs) were also developed to evaluate the cell apoptosis induced by 2-BP/CPT-PLNs as previous report [34 (link)]. In brief, B16-F10 cells were seeded onto the U-bottom ultra-low adsorption microplates culture plate at 1000 cells/well to yield B16F10 MCTs. Then these MCTs were treated with CPT, CPT + 2-BP, or 2-BP/CPT-PLNs for 24 h. After removing the old medium containing drugs, the resulting MCTs were immediately stained with Hoechst 33,342 and PI (Beyotime), and imaged with CLSM.

For apoptosis analysis, EC cells were incubated with 10 µm of JX57 for 48 h. Following the collection of the cell supernatant, cells were trypsinized and centrifuged. Harvested cells were then washed twice with PBS and stained for 15 min with Annexin V‐FITC/PI (Yeasen). The stained cells were evaluated using flow cytometry (BD Bioscience, Franklin Lakes, NJ, USA).