Fitc labeled anti human cd14

CD14⁺ monocytes were isolated as described before (ten Harkel et al., 2015). Briefly, peripheral blood mononuclear cells were isolated from human buffy coats from healthy donors (Sanquin, The Netherlands) using Ficoll‐Paque density gradient centrifugation. Subsequently, cells were incubated with CD14‐antibody tagged microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and sorted using a manual MACS magnetic cell sorter (Miltenyi Biotec) according to the manufacturer's instructions (Davison et al., 2014). The purity of the cells was determined with flow cytometry (FACSverse^™; BD Biosciences, Piscataway). For analysis, cells were incubated with fluoresceine isothiocyanate (FITC) labeled anti‐human CD14 (Miltenyi Biotec) or its equivalent isotype control IgG2a (Miltenyi Biotec), and incubated for 30 min in the dark on ice. After incubation, cells were washed to remove unbound antibodies, recovered in FACS buffer and analyzed (30 s or 100,000 viable events) on a BD Bioscience FACSverse flow cytometer. Purity was confirmed to be at least 80%.

For the initial TRAcP staining and QPCR experiments CD14⁺ monocytes were isolated from the blood from 6 FOP patients and sex- and age- matched controls (20–40 ml blood per donor), for all other experiments human buffy coats (Sanquin, The Netherlands) or blood from healthy donors was used. The CD14+ cells were isolated as described before (23 (link)).
Briefly, peripheral blood mononuclear cells were isolated using Ficoll-Paque density gradient centrifugation. Subsequently cells were incubated with CD14-antibody tagged microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and sorted using a manual MACS magnetic cell sorter (Miltenyi Biotec) according to the manufacturer's instructions (24 (link)). The purity of the cells was determined with flow cytometry (FACSverse™, BD Biosciences, Piscataway, USA). For the analysis, cells were incubated with FITC labeled anti-human CD14 (Miltenyi Biotec) or its equivalent isotype control IgG2a (Miltenyi Biotec) for 30 min in the dark on ice. After incubation, cells were washed to remove unbound antibodies, recovered in FACS buffer and analyzed (30 s or 100,000 viable events) on a BD Bioscience FACSverse flow cytometer. Purity was confirmed to be at least 80%.