Cd13 phycoerythrin cyanine 7 pe cy7

Phagocytosis of apoptotic cells was measured as previously reported [3 (link),4 (link),30 (link)]. Briefly, 16HBE bronchial epithelial cell targets were maintained in continuous culture, induced to apoptosis using UV, then labelled with sytox orange (Molecular Probes, Oregon, USA). The apoptotic cells were incubated with macrophages at a ratio of 10:1 for 1.5h. Non-adhered cells were removed and macrophages removed by gentle pipetting, following 5min incubation with 500uL ice-cold phosphate buffered saline (PBS). Macrophages that had ingested apoptotic cells were stained with CD13 phycoerythrin cyanine-7 (PE-Cy7) (BD Biosciences, San Jose CA, USA)), autofluorescence was quenched with trypan blue and 30,000 total events per tube were acquired immediately using a FACSCanto II Flow Cytometer (BD Biosciences). Macrophages were identified based on autofluorescence properties and staining with CD13. The percentage of the macrophages ingesting apoptotic cells was recorded.
To further elucidate functional effect of increased S1PR5 on macrophage phagocytic ability, we performed the phagocytosis assay in the presence of varying concentrations of Suramin (Sigma Aldrich, Castle Hill, NSW, Australia), an antagonist of S1PR3 and S1PR5 [31 (link)]. Suramin at concentrations of 10nM to 10μM was added for 30min prior to the phagocytosis assay.

For efferocytosis and phagocytosis assay: CD13 phycoerythrin cyanine-7 (PE-Cy7) (BD Biosciences, San Jose CA, USA), sytox orange and sytox green (Molecular Probes, Oregon, USA) were employed. For other flow cytometry staining: mannose receptor phycoerythrin (PE) (Immunotech/Coulter, Marseille, France); Colec-12 unconjugated (R&D Systems, Minneapolis, MN, USA), detected with anti-goat IgG allophycocyanin (APC) (R&D) and, CD204 PE (R&D) were employed. For immunofluorescence: MSR1/SRA unconjugated rabbit polyclonal antibody (Abcam, Cambridge, UK and sheep anti-rabbit IgG-Cy3, F(ab’)2 fragment (Sigma-Aldrich, Saint Louis, MO, USA) was employed. All other reagents, if not otherwise specified, were from Sigma-Aldrich (Saint Louis, MO, USA).