BM-MNCs from 5 BCPs and 8 HVs, as well as PB-MNCs from 5 BCPs and 5 HVs, were collected and isolated as described above. Viable MNCs (5 x 105) were incubated in 5% AB serum (S7148, Sigma) diluted in PBS for 30 min at 4°C, to reduce non-specific binding. Single labelling using primary Abs anti-human: anti-RANK (RANKL receptor, mouse IgG1, MAB683, R&D Systems), anti-CD11b-PE (mouse IgG2ak, 347557, BD Bioscience), anti-CD14 (mouse IgG1k, 14-0149, eBioscience), anti- CD51/61-FITC (integrin alphaV/beta3, mouse IgG1k, 555505, BD Bioscience) and anti-CD115 (or c-fms, M-CSF receptor, rat IgG1, 4-1159, eBioscience) were performed. For uncoupled Abs an incubation with secondary Ab FITC-conjugated (anti mouse-IgG, 115-095-164, Jackson Inmuno Research) or PE-conjugated (anti rat-IgG-PE, F0105B, R&D Systems) followed. Isotype controls were run in parallel using the same concentration of each primary Ab tested. Isotype controls: mouse IgG1 (MAB002, R&D system), mouse IgG2ak-PE (556653, BD Bioscience), mouse IgG1k (ab91353, abcam), mouse IgG1k-FITC (551954, BD Bioscience) and rat IgG1 (14-4301-82, eBiosciences). After labelling, cells were washed using 3% BSA (A7906, Sigma) in PBS (BSA-PBS). Thereafter, cells were fixed in 1% formaldehyde (1044, Cicarelli Corp.) in PBS for 20 min at 4°C. At least 10,000 events were acquired using FACScalibur (BD Biosciences). FlowJo X v10.0.7 (FlowJo, LLC) software was used to create the histograms and density plots. Results were expressed as percentage and relative fluorescence index (RFI= marker mean fluorescence index/corresponding isotype control mean fluorescence index). Experiments were repeated two times for each individual sample.
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