Flow cytometry and immunohistochemical characterization of bone marrow-derived CD115+ monocytes

Bone marrow cells were harvested from young donor wild-type mice (n = 3–4 mice per experiment) as detailed above. After enrichment for mononuclear cells by Ficoll® gradient, one portion of cells was collected (before CD115⁺ column), and a second portion of cells underwent further isolation for CD115⁺ monocyte population (after CD115⁺ column), using the MACS enrichment column as detailed above. Both cell portions, before and after CD115⁺ column, were either immediately stained and analysed by flow cytometry or plated to generate macrophage cultures for further immunohistochemical analysis (see Supplementary Fig. 4). For flow cytometry analysis, the before CD115⁺ column cells were stained with the following antibodies: biotinylated anti-CD115 mAb clone AFS98 (#13-1152; eBioscience), APC-conjugated anti-Biotin clone Bio3-18E7 (#130-090-856; Miltenyi Biotec), PE-conjugated anti-CD36 clone REA262 (#130-102-763; Miltenyi Biotec), Viobright FITC-conjugated anti-CD36 clone REA262 (#130-104-889; Miltenyi Biotec), PE-conjugated anti-CD204 clone REA148 (#130-102-328; Miltenyi Biotec), and Alexa Fluor® 488-conjugated anti-MMP9 polyclonal antibody (#bs-0397R-A488; Bioss). For the after CD115⁺ column cells, we used a set of staining antibodies identical to that specified above but excluded the primary anti-CD115 mAb because this isolation procedure already linked the biotinylated anti-CD115 antibody to the cells. All antibody dilutions were 1:100. The labelled samples were analysed on a BD LSRFortessa™ Cell Analyzer equipped with BD FACS Diva software; data were further analysed with FlowJo software (vX.0.7r2; Tree Star, Inc.).

Both cell portions, before and after CD115⁺ column selection, were also differentiated into primary macrophage cultures and analysed by immunohistochemistry. In brief, cells were differentiated into macrophages by 7-day cultivation in complete RPMI-1640 medium (#21870; Life Technologies) with 10% serum and 20 ng/ml MCSF (#315-02; PeproTech). Primary cultures of macrophage were then plated at 1.2 × 10⁵ cells per well (3–4 wells for each condition) in 24-well tissue-culture plates on glass coverslips overnight. Methanol (99.8%) at −20°C for 20 min was used for fixation of the cells followed by repeated washes with PBS. Cells were then stained using rat anti-CD36 mAb clone MF3 (1:200; ab80080; Abcam), rat anti-CD204 scavenger receptor type I/II (SCARA1) mAb (1:100; MCA1322; AbD Serotec), and goat anti-MMP9 pAb (1:100; AF909; R&D systems). Secondary polyclonal antibodies included donkey anti-rat and anti-goat conjugated with Cy2, Cy3 or Cy5 (1:200; Jackson ImmunoResearch Laboratories). The cells were mounted using ProLong® Gold with DAPI (Molecular Probes, Life Technologies). Several fields (minimum n = 4 randomly selected per group) were obtained from each well using a Carl Zeiss Axio Imager Z1 Apotome-equipped microscope (an average of 120 cells in each field). Images were obtained using the same exposure time in each occasion. The fluorescent signal and its total area were determined and quantified by the conversion of the individual images to grey scale and standardizing to baseline using histogram-based thresholds with NIH ImageJ software. The ‘area/cell’ measures the total fluorescent signal (area) divided by the total number of cells (DAPI count) of the same field (image). For all experiments, the investigators were blinded to the treatment condition.