Megafasl

Resident and thioglycollate-elicited peritoneal cells, peritoneal macrophages, bone marrow-derived monocytes, brain microglial cell, and photoreceptor outer segment (POS) isolation, and MP-retinal explant co-cultures (all in serum-free X-VIVO 15 medium) were performed as previously described (Sennlaub et al, 2013 (link)). In specific experiments, cells were stimulated with recombinant human CX3CL1 or APOE3 (5 μg/ml, Leinco Technologies), APOE3 (5 μg/ml) with polymyxin B (25 μg/ml, Calbiochem), heat-denatured APOE3 (5 μg/ml, 95°C, 90 min), rat anti-IgG isotype control (100 μg/ml, R&D), rat anti-mouse CD14 (100 μg/ml, R&D), mouse anti-mouse TLR2 (100 μg/ml, Invivogen), and POS prepared as previously described (Molday et al, 1987 (link)). For in vitro apoptosis experiments, 100,000 Mos or Mϕs of the different genotypes were cultured for 24 h with or without MegaFasL (1 ng/ml, AdipoGen). TUNEL staining (In Situ Cell Death Detection Kit, Roche Diagnostics) was performed according to the manufacturer's instructions; TUNEL⁺ and Hoechst⁺ nuclei were counted automatically using the Array Scan (Thermo Fischer).

Ultra-pure flagellin (Psuedomonas aeruginosa), Pam3CSK4, R837 (Imiquimod), ultra-pure LPS (E. coli O111:B4), Poly(dA:dT), Nigericin were purchased from Invivogen, Mega FasL from AdipoGen, IFN-α and IFN-β from PBL Assay Science, TNFα from Genentech, ATP and bovine cytochrome-c from Sigma, YOYO-1 dye from Thermofisher Scientific, Nuclear-ID DNA stain from Enzo Life Sciences. Antibodies used include: mouse caspase-1 (clone 4B4, Genentech), Gsdmd (17G2G9, Genentech), caspase-3 (Cell Signaling Technology), cleaved active caspase-3 (5A1E, Cell Signaling Technology), caspase-8 (1G12, Enzo), cleaved active caspase-8 (D5B2, Cell Signaling Technology), IL-1β (GTX74034, GeneTex) and actin (AC15, NOVUS) and DFNA5 rabbit polyclonal antibody was raised against ESDFVKYESKCENHKSGAIG peptide (mouse DFNA5 74–93 amino acid) and FLAG M2 antibody (Sigma).