Annexin 5 pi apoptosis detection kit 1

Annexin V/PI Apoptosis Detection Kit I (BD Biosciences Pharmingen, USA) was used to determine the cell apoptosis. Briefly, 30,000 cells were collected and washed in a 2 mL of cold PBS. Then, 100 mol/L of 4X combined buffer was mixed on the ice. Annexin V 5 ul and Propidium Iodide 5 ul (1 mg/mL) were added, and the cells were incubated on ice for 15 min in the dark. The samples were then diluted with a 4 × 400 μl binding buffer. Cell analysis was performed using flow cytometry (Becton Dickinson, USA) and FlowJo Version 8.6 (Treestar Inc., San Carlos, CA).

Cell proliferation assays were performed using Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies #CK04-11) as previously reported.^{54 (link),56 (link),63 (link)} Briefly, the parental and resistant cells (1.5 × 10⁴) in RPMI-1640 medium (100 µl) were dispensed into 96-well flat-bottomed microplates and drugs were added after 24 h of incubation. The cells were cultured for another 24 or 48 h, and CCK-8 (10 µl) was added to each well. The microplates were incubated at 37 °C for additional 2~4 h. Absorbance was read at 450 nm using a microplate reader and the results were expressed as a ratio of the treated over untreated cells (as 100%). Four wells were sampled per each experimental group in a given experiment. Averages are reported ± SD.
Cell apoptosis assays were performed using Annexin V-PI Apoptosis Detection Kit I (BD Pharmingen^TM #556547) according to the manufacturer’s instruction, and followed by flow cytometry analysis. The EdU incorporation assays were performed using Click-iT™ EdU Alexa Fluor™ 647 Flow Cytometry Assay Kit (ThermoFisher #C10419) according to the manufacturer’s instruction.