Acoustic Separation of Neuroblastoma Cells

The acoustic separation of neuroblastoma cells from CD34⁺/PBPCs was performed as previously described [17 (link)]. Optimal actuation frequencies and voltages for the prealignment and separation channel transducers were initially determined in calibration experiments by separation of 5 μm and 7 μm polystyrene microspheres (Sigma-Aldrich). Separation performance was analyzed by flow cytometry (FACS Canto II, BD Biosciences, San Jose, CA, USA). PDX cells and PBPC samples were labelled with directly fluorochrome-conjugated monoclonal antibodies CD45-FITC (clone 2D1), CD34-PE (clone 581), and CD56-APC (clone B159, all BD Bioscience) prior to acoustic sorting. Propidium iodide (Sigma-Aldrich) was used for dead cell exclusion. PBPCs were identified as CD45⁺, and CD34⁺ cells as CD45^low/CD34⁺ along with scatter properties in accordance with the ISHAGE guidelines [19 ]. Neuroblastoma PDX cells were identified as CD45⁻/CD56⁺. Data were analyzed using FlowJo v10 software (FlowJo LLC, Ashland, OR, USA). Relative recovery rates were calculated using the Additional file 1: Equations 3.