Human macrophage efferocytosis of senescent erythrocytes and neutrophils
This protocol is extracted from research article:
Resolution metabolomes activated by hypoxic environment
Sci Adv, Oct 23, 2019; DOI: 10.1126/sciadv.aax4895

Human erythrocytes were kept in DPBS containing Ca2+ and Mg2+ at 20% ht at 4°C for 24 hours to induce apoptosis. RBC senescence was assessed by diluting 1 μl of human whole blood or senescent cells into 100 μl of fluorescence-activated cell sorting (FACS) staining buffer followed by staining with anti-human CD235a allophycocyanin (APC)–Cy7 (clone HI264) and anti-human phycoerythrin (PE) CD47 (clone CC2C6) for phenotyping. RBCs were then suspended at 10% ht and stained with carboxyfluorescein succinimidyl ester (CFSE; 5 μM, 30 min, 37°C) and were washed three times with phosphate-buffered saline (PBS) via centrifugation and aspiration of supernatant. Human neutrophils were suspended in DPBS containing Ca2+ and Mg2+ (5 × 107 cells/10 ml) and placed in petri dishes and incubated for 24 hours at 37°C to induce apoptosis. Apoptotic neutrophils were removed from petri dishes with EDTA (5 mM) and stained with CFSE (5 μM, 30 min, 37°C). Human M2 macrophages were seeded in six-well plates (2 × 106 cells per well), treated with RvD5, RvD6, or RvE4 (10 nM), SPM panel (RvD2, RvD5, RvD6, MaR1, PD1, and RvE4; each together at 10 nM), or vehicle (0.01% ethanol) 15 min before addition of CFSE-labeled erythrocytes [1:50 (M2:RBC) ratio] or CFSE-labeled neutrophils [1:5 (M2:neutrophil) ratio]. After 1 hour of coincubation at 37°C, plates were washed thoroughly to remove noningested RBC, and cells were fixed with PBS containing 4% paraformaldehyde for 15 min on ice. Cells were washed with DPBS and removed from plates with a cell scraper in DPBS. Cells were then taken to flow cytometry to assess efferocytosis.

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