Apoptosis-Mediated TREM2 Activation in Neurodegeneration

Phosphatidylserine (PS) expression on the external surface of apoptotic SH-SY5Y cells was used as a ligand for TREM2 as we previously reported^{10 (link)}. Briefly, SH-SY5Y cells (a kind gift from Prof R de Silva, University College London, Queen Square Institute of Neurology) were cultured in DMEM with 10% FBS (Life Technologies) and 1% penicillin/streptomycin (Life Technologies). To induce apoptosis, SH-SY5Y cells were heat shocked at 45 °C for 2 h, before resuspension in iPS-Mg medium. Cell death and exposure of cell membrane PS was confirmed by flow cytometry using FITC-conjugated Annexin-V (Miltenyi Biotec) and propidium iodide staining. Apoptotic cells (“PS+ cells”) were added to iPS-Mg at a ratio of 2:1 PS+ cells:iPS-Mg for either 5 min (signalling assays) or O/N (inflammasome assays). Exposure of iPS-Mg to PS+ cells was blocked by preincubation of PS+ cells for 1 h with 5 µg recombinant Annexin V (268–10,001, RayBiotech). SYK signalling was blocked by preincubation of iPS-Mg for 1 h with 10 µM BAY61-3605 prior to addition of PS+ cells.