Annexin 5 pi staining assay kit

Cells were incubated with KPT‐330 (100 nm) and/or Y219 (6 nm) for 48 h. Cellular DNA content was quantified using PI/RNase Staining Buffer (BD Biosciences, San Diego, CA, USA). Cell apoptosis was detected using the Annexin V/PI staining assay kit (BD Biosciences). Cell cycle and apoptosis were analyzed by flow cytometry.

Cell viability assay was determined using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). In brief, cancer cells were suspended and seeded in the culture medium in a 96-well plate. After 24 h, cells were treated with the compounds at 37 °C for 1 h or 72 h followed by washing thrice with PBS and further incubation in fresh medium for an additional 72 h. The CCK8 reagent was added to each well and the plates were incubated at 37 °C for another 1-2 h. The absorbance was measured with a microplate reader (CLARIOstar; BMG Labtech) at 450 nm. Cell viability was also assessed by exclusion of trypan-blue staining. The viabilities of CD138-positive cells and PBMCs were assessed using the Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega, Corp., Madison, WI, USA) with cells plated and processed as described above. Cellular DNA content was quantified using a propidium iodide (PI) staining assay kit (BD Biosciences, San Diego, CA, USA). The percentage of apoptotic cells was detected using the Annexin V/PI staining assay kit (BD Biosciences, San Diego, CA, USA). Cell cycle and apoptosis were analyzed by flow cytometry (FACSCalibur, BD Biosciences).