Annexin 5

Fixed right humeri were decalcified in 14% EDTA (~14 days) and embedded in paraffin. Serial 5 μm sections were taken. Sections were stained utilizing a standard avidin-biotin method as previously described [9 (link)]. Samples were stained for receptor activator of nuclear factor κB ligand (RANKL; Abcam, Cambridge, MA, USA), annexin V (Abcam), an early marker of cellular apoptosis, and two proinflammatory cytokines, tumor necrosis factor-α (TNF-α; Abcam) and interleukin-17 (IL-17; Abcam). Peroxidase development was performed with an enzyme substrate kit (DAB, Vector Laboratories, Burlingame, CA, USA). Counterstaining was conducted with methyl green (Vector Laboratories) which stain osteocyte nuclei. Negative controls for all antibodies were completed by omitting the primary antibody. Sections of the entire humeral midshaft cortical bone in cross-section were analyzed and results are reported as the percentage of osteocytes stained positively for the protein (DAB-positive) relative to all osteocytes (DAB-positive and methyl green-positive) in the cross-section. All analyses were completed by the same individual.

GFP expressing LR73 fibroblasts were seeded in a 24-well plate at a density of 0.2×10⁵ cells per well. Next day, cells were treated with Lipofectamine 2000 (Thermo Fisher) with specific siRNAs according to the manufacturer’s protocol, two days prior to efferocytosis assays. Thymocytes were isolated from 4 to 6-week-old mice and treated with 25 μM dexamethasone for 4 h to induce apoptosis. Apoptotic thymocytes were then stained with 10 μM TAMRA in serum-free HBSS for 45 min at 37 °C, washed, and incubated in serum-containing assay media for an additional 25min. Phagocytes were pre-incubated with Cytochalasin D (Sigma-Aldrich, 1 μM) for 1 h and, in some experiments, Tritbutyltin chloride (Sigma; 10 μM for 6 h), or with RGDS peptide for 30 min (Tocris 100 μM); in some cases apoptotic cells were incubated with Annexin V (Abcam, 10 μg/ml for 30 min). Apoptotic cells were subsequently co-cultured with phagocytes, in the presence of vehicle or indicated inhibitor, at a 1:10 phagocyte to target ratio for 30 minutes. After the incubation, apoptotic cells were removed via three cold PBS washes and phagocytes were harvested via trypsin/EDTA and assessed for the percentage and MFI of GFP⁺ TAMRA⁺ LR73 cells by flow cytometry.