All experiments containing SnPP or hemin treatment were conducted in the dark with a dim light to minimize inactivation p38 MAPK of these compounds. Cell culture plates or petri dishes were kept in a dark box Inhibitors,Modulators,Libraries to prevent light exposure. Cell viability assay To determine the effect of hemin or SnPP on astrocyte viability Inhibitors,Modulators,Libraries a MTT assay, which provides quantitative assessment of mitochondrial integrity, was used. After treatment of astrocytes with hemin or SnPP, MTT was added to cell cultures for 4 h followed by addition of lysis buffer for 16 h. Cell lysate was collected and absorbance was read at 600 nm to reflect possible cytotoxicity caused by treatment. Another cell proliferation and cytotoxicity assay using alamarBlue, in which the living cells convert the non toxic, cell permeable and non fluorescent resazurin to red fluorescent resorufin, was measured at Ex 560 nm and Em 590 nm to verify cell viability.
Enzyme linked immunoabsorbent assay After treatment, astrocyte culture supernatants were col lected for ELISA measurement of cytokines Inhibitors,Modulators,Libraries and chemokines. In brief, 96 well ELISA plate pre coated with mouse anti human cytokinechemokine antibody overnight at 4 C was blocked with 1% BSA in PBS for 1 h at 37 C. After washing with PBS with Tween 20, culture supernatants and a series of dilution of cytokineschemokines were added to wells for 2 h at 37 C. Goat anti human cytokinechemo kine detection antibody was added for 90 min followed by addition of donkey anti goat IgG horseradish peroxi dase conjugate for 45 min. A chromogen sub strate K Blue was added at room temperature for color development which was terminated with 1 M H2SO4.
The plate was read at 450 nm and cytokinechemokine concentrations were extrapolated from the standard concentration curve. NO assay After treatment, astrocyte culture supernatants were col lected to measure nitrite release using Griess Inhibitors,Modulators,Libraries reagent, which reflects NO production in cultures as previously described. In brief, Griess reagent consisting of equal volumes of 0. 1% naphthylenediamine dihydrochloride in distilled H2O and 1% sulfanilamide and 6% H3PO4 in distilled H2O, was added in equal volume Inhibitors,Modulators,Libraries to astrocyte culture supernatants. After 10 min incubation at room tempera ture, the mixtures were read with a microplate reader at 550 nm and NO2 level was extrapolated from a stan dard curve generated with a series of concentrations EPZ-5676 buy of sodium nitrite. The detection limit for NO2 was 0. 5 uM. iNOS immunoassay To determine the iNOS concentration in cell lysates, astrocyte cultures were untreated or pretreated with hemin for 24 h prior to IL 1b treatment for 72 h. Cell lysates were collected and assayed accord ing to manufacturers protocol.