In vitro efferocytosis assay (Flow cytometry)

Macrophages were differentiated in vitro from iPSC derived progenitors or PBMC monocytes as described above. In brief 5 × 10⁴ cells/well were cultured in culture medium (X-VIVO15, 1% P/S, 2 mM Glutamax) with indicated stimuli for seven days in 96-well flat bottom plates. Jurkat cells were maintained in RPMI1640 (2 mM Glutamax, 10% FBS, MEM nonessential aminoacids). Apoptosis was induced by culturing cells at 1 × 10⁶ cells/ml Jurkat medium containing 2.5 µM staurosporine (Sigma) at 37 °C with 5% CO₂ for 3 h. For live cell controls, Jurkat cells were cultured with addition of solvent (DMSO) under indicated conditions. Afterwards, cells were labeled with pHrodo Red SE (Invitrogen) according to the manufacturer protocol and washed twice with PBS + 1% BSA + 1 mM EDTA) and once with PBS. Apoptosis induction was verified by flow cytometry using the AnnexinV-Fitc staining kit (BD) with fixable viability dye-eFluor780 (eBioscience). For some conditions apoptotic cells were labeled with 50 nM recombinant murine GAS6 (Roche, internally produced) for 15 min in iPSC medium at RT. Apoptotic or live cells were added to macrophages at a ratio of 6:1 (Apoptotic cells: macrophages) in culture medium for 2 h. For some experiments macrophages were incubated with anti-Axl (AF154, R&D), anti-MERTK (AF891, R&D), or control antibody serum (AB-108-C, R&D) at 5 µg/ml for 30 min. Similarly, small molecule inhibitors for AXL (R428, Selleckchem, Zürich, Switzerland) or MERTK (UNC569, Merck, Darmstadt, Germany) or solvent control (DMSO) were added for 30 min to the cells at a concentration of 20 nM. Macrophages were washed once with PBS, detached with Accutase, stained with fixable viability dye in eFluor780 (eBioscience), and analyzed by flow cytometry.