Flow cytometric data analysis software

SKO-007(J3) cells or SKO-007(J3)/shMEIS2-Tet cells were cultured in 6-well tissue culture plates at a concentration of 2 × 10⁵ cells/ml in the presence of the indicated drug(s). The expression of the NKG2D and DNAM-1 ligands on MM cells was analyzed by immunofluorescence staining using unconjugated mAbs, followed by secondary GAM-APC. In all experiments, cells were stained with Propidium Iodide (PI) (1 µg/ml) in order to assess cell viability (always higher than 90% after the different treatments). Nonspecific fluorescence was assessed by using an isotype-matched irrelevant mAb R&D System, followed by the same secondary antibody. Fluorescence was analyzed using a FACSCalibur flow cytometer BD Biosciences and data were analyzed using FlowJo Flow Cytometric Data Analysis Software (FlowJo - Ashland, OR). The analysis of ligand expression on patient derived plasma cells was performed by gating on the CD138⁺ and CD38⁺ PC population.

Apoptotic cell death was evaluated using APC Annexin-V Apoptosis Detection Kit (BioLegend). Briefly, MM cell lines or purified CD138⁺cells from patients, were cultured in six-well tissue culture plates at 2 × 10⁵ cells/ml (or 1 × 10⁶ cells/ml for patient’s MM cells), untreated or treated with MLN4924 for 72 h as described above. Cells were then stained using Annexin-V/APC according to the manufacturer’s instruction. Flow cytometric analysis was performed using a FACS Canto II flow cytometer (BD Biosciences) and data were analyzed using FlowJo Flow Cytometric Data Analysis Software (FlowJo—Ashland, OR, USA).