Cd56 apc clone b159

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.

Freshly isolated cells were stained with relevant antibodies for a total of 20 min at room temperature and analyzed by flow cytometry. Due to various sample amounts (number of cells in each sample), we were not able to perform all analyses on all samples and patients. The antibodies (with dilutions in parenthesis) used were as follows: CD14-fluorescein isothiocyanate (FITC) clone M5E2 (1:10), HLA-DR-phycoerythrin (PE) clone G46-6 (1:20), HLA-DR-allophycocyanin (APC) clone L243 (1:50), CD86-PE clone IT2.2 (1:20), CD33-APC clone WM53 (1:10), CD3-FITC clone HIT3a (1:25), CD4-APC clone RPA-T4 (1:25), CD8-PE HIT8a (1:25), CD8-APC clone RPA-T8 (1:25), CD25-FITC clone 2A3 (1:10), CD127-PE clone HIL-7R-M21 (1:20), CD45RA-FITC clone HI100 (1:10), CD45RO-PE clone UCHL1 (1:10), CD19-PE clone HIB19 (1:20), CD138-APC clone MI15 (1:15), CD183-PE clone 1C6/CXCR3 (1:25), EpCAM-FITC clone EBA-1 (1:25), CD66b-PE clone G10F5 (1:20), CD64-APC clone 10.1 (1:20), CD294-FITC clone BM16 (1:25), CD209-APC clone DCN46 (1:15), CD203c-PE clone NP4D6 (1:25), CD196-PE clone 11A9 (1:20), CD161-FITC clone DX12 (1:10), and CD56-APC clone B159 (1:10), all from BD Biosciences. Cells were analyzed using an FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). All analyses were performed using 7-AAD dead exclusion stain (BD Biosciences).