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Cells were washed three times with ice-cold PBS and lysed in lysis buffer (40 mM Tris-HCl (pH 8.0), 1% Triton X-100, 1% NP-40, 10% glycerol, 0.15 M NaCl, 2 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, 1 Complete protease inhibitor cocktail)

As shown in an earlier publication, unconjugated bilirubin stimulates microglia primary cultures to respond by acquiring a phagocytic phenotype, which is transformed into an inflammatory response featuring secretion of the pro-inflammatory cytokines interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-, increased regulation of cyclooxygenase (COX)-2, and enhanced the activities of MMP- 9 and -2 (188)