Anti cd45 pc5

Blood was centrifuged and serum was stored in -80 °C until assayed. Levels of biomarkers were quantified by using commercially available kits of enzyme immunosorbent assays from Bio-Techne (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. The lower limits of detection were: 16 pg/ml for tumor necrosis factor-alpha (TNFα); 40 pg/ml for IL-6; 31 pg/ml for IL-10; 62 pg/ml for IL-38; 156 pg/ml for IFNγ; and 313 p/ml for platelet-derived growth factor (PDGF)-A.
White blood cells were incubated for 15 minutes in the dark with the monoclonal antibodies anti-CD14 FITC, anti-HLA-DR-PE, anti-CD45 PC5 (Beckman Coulter, Marseille, France). White blood cells were also incubated for 15 minutes in the dark with anti-CD3 FITC, anti-CD4 FITC and anti-CD19 FITC (fluorescein isothiocyanate, emission 525nm, Beckman Coulter); with anti-CD4 PE, anti-CD8 PE, and anti-CD(16+56) PE (phycoerythrin, emission 575nm, Beckman Coulter); and with anti-CD45 PC5 (emission 667nm, Beckman Coulter). Fluorospheres (Beckman Coulter) were used for the determination of absolute counts. Cells were analyzed after running through the CYTOMICS FC500 flow cytometer (Beckman Coulter Co, Miami, Florida). Isotypic IgG controls stained also with anti-CD45 were used for each patient. Results of HLA-DR on CD14/CD45-cells were expressed as mean fluorescence intensity (MFI).

The myogenic nature of purified cells was evaluated by flow cytometry analysis performed on CytoFLEX (Beckman Coulter) as previously described (Awaya et al., 2012 (link); Lapan and Gussoni, 2012 (link)); the following panel of antibodies was used to determine immunophenotype: anti-CD56 PC7 (Beckman Coulter, USA, A21692), anti-CD146 PE (Beckman Coulter, USA, A07483), anti-CD166 PE (Beckman Coulter, USA, A22361), anti-CD73 PE (BD Pharmingen, USA, 550257), anti-CD105 APC (R&D Systems, USA, FAB1097A-100), and anti-CD45 PC5 (Beckman Coulter, USA, A07785). Data were analyzed using the CytExpert 2.0 (Beckman Coulter). The phenotypic characteristics of the obtained cells are illustrated in the Supplementary Material, Figure S2A.

Flow cytometer analysis was performed on whole BM samples, using 50 μl of whole blood/tube. Briefly, samples were incubated with specific antibodies (20′ at 4°C in the dark) and then subjected to erythrocytes lysis using BD FACS lysis (BD Biosciences, Milan, Italy) by incubating 15′ at RT in the dark. Cells were washed with PBS 0.5% BSA, suspended in PBS 0.5% BSA and run on Gallios cytometer (Beckman Coulter, Cassina dei Pecchi, Italy), acquiring at least 10⁴ events. Data were analyzed using Kaluza software (Beckman Coulter). The antibodies used were: anti-CD45 PC5 (Beckman Coulter), anti-CD35 FITC (Immunotools, Friesoythe, Germany), anti-CD44 PE (Immunotools), anti-CD117 APC (Miltenyi Biotec, Calderara di Reno, BO, Italy), anti-CD16 PC7 and anti-CD14 FITC (Beckman Coulter), following manufacturer's protocol. PB from 11 NB patients and 10 controls were analyzed for monocytes and granulocytes, whereas PB from 15 NB patients and 10 controls were analyzed for stage II and III erythroblasts.