Cd45 fitc clone 2d1

Staining of unfractionated fresh blood was performed according to the lyse and stain approach using monoclonal antibodies: CD45 FITC (clone 2D1), CD4 PE/CF594 (clone RPA-T4, BD Biosciences, San Jose, CA, USA), CD3 PE-Cy7 (clone UCHT1), CD8 BV510 (clone SK1), CD16 PE (clone B73.1), HLADR BV786 (clone L243, BioLegend, San Diego, CA, USA) and the results read in the Fortessa flow cytometer (BD Biosciences, San Jose, CA, USA). Living cells (stained with LIVE/DEAD Fixable Dead Cell Stain Kit with BV421 fluorochrome, Life Technologies Carlsbad, CA, USA) were evaluated. The gating was done using both CD45 and side scatter signals. NovoExpress Software (Agilent Santa Clara, CA, USA) was used for subpopulation analysis.

100 µl of blood sample are stained with 5 µl of each antibody for 30 minutes in the dark against CD34-PE (clone 8G12; BD Biosciences, Heidelberg, Germany) CD45-FITC (clone 2D1; BD Biosciences, Heidelberg, Germany) and the viability dye 7AAD (Beckman Coulter Inc., France) applying the CytoFlex flow cytometer (Beckman Coulter Inc., France). Using a lyse/no-wash approach for whole-blood samples, 1 mL of Versa Lyse solution (Beckman Coulter Inc., France) is added and incubated for 15 minutes at room temperature in the dark. Determination of CD34⁺ cells was performed according to the guidelines provided by the International Society of Hematotherapy and Graft Engineering (ISHAGE-protocol).^{20 (link)}

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.