Tibolone

Astrocytes were incubated for 6 h in DMEM with 10% FBS and then 15 h with the following test compounds, either alone or in combination in DMEM 0.1% FBS: tibolone (100 nM; Sigma-Aldrich); ERα antagonist 1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidyletoxy) phenol]-1H-pyrazole (MPP, 100 nM; Sigma-Aldrich); the ERβ antagonist 4-[2-phenyl-5,7-bis (trifluoromethyl) pyrazolo [1,5-a] pyrimidin-3-yl] phenol (PHTPP, 100 nM; Tocris, Bristol); the G-protein-coupled ER (GPER) antagonist (3aS,4R,9bR)-4-(6-bromo-1,3-benzodioxol-5-yl)-3,4,5,9B-tetrahydro-3H-cyclopenta[c]quinolone (G15, 100 nM; Tocris); the androgen receptor antagonist flutamide (100 nM; Tocris); or the aromatase inhibitor letrozole (100 nM; Tocris). Twenty-four hours before phagocytosis assay astrocytes were incubated with DMEM serum-free medium with the previous compounds plus lipopolysaccharide (LPS) isotype O26:B6 (1 μg/ml; Sigma-Aldrich, Tres Cantos, Madrid). The concentrations used for the test compounds were chosen on the basis of previous studies [11 (link), 42 (link)]. After the incubation with the test compounds, phagocytosis activity was assessed as indicated below.

Cells were pre-treated with Tibolone prior to the addition of PA. Tibolone (Lot T0827, Sigma, St Louis, MO, USA) was dissolved in DMSO as a stock solution at 40 mM, and further dilutions were prepared with serum-free DMEM to a final concentration of 0.000025%. Different times and concentrations of Tibolone treatment were tested, and 10 nM of Tibolone for 24 h was found to best preserve cell viability upon PA treatment.