Although several signaling pathways are activated by IL 1b in astrocytes, we focused on mitogen activated protein kinases to determine if the effect of hemin on IL 1b stimulated astrocytes is mediated through a MAPK signaling path way. We also looked into the possible effects of hemin on IL 1b stimulated selleck MEK162 cytokine and chemokine production to assess whether HO 1 also dampens the production Inhibitors,Modulators,Libraries of these inflammatory mediators. Methods Reagents The following reagents were purchased from the indi cated sources hemin and Sn Protoporphyrin IX dichloride 3,7,12,17 tetramethyl 21H,23H porphine 2,18 dipropionic acid tin dichloride. IL 1b, tumor necrosis factor a, CXCL10, Human iNOS Quantikine ELISA Kit, anti human TNF a and CXCL10 antibodies. anti p38 and extracellular signal regulated kinase 1 and 2 MAPK antibodies.
SB203580 and U0126. mouse anti HO 1 anti body. RNase inhibitor, SuperScript III reverse transcriptase Inhibitors,Modulators,Libraries and alamarBlue. DNase. oligo 12 18. Inhibitors,Modulators,Libraries SYBR Premix Ex Taq. SYBR Advan tage qPCR premix. dNTPs. rabbit anti NOS2 and HO 2 antibodies. rabbit anti GFAP. LentiORF pLEX MCS vector. Fugene 6. M PER. Dulbeccos modified Eagles medium, bovine serum albumin, and 3,3 diaminobenzidine, Inhibitors,Modulators,Libraries 3 2,5 diphenyl 2H tetrazolium bromide. acrylamidebis acrylamide gel and protein assay. CDP Star substrate. K Blue substrate. heat inactivated fetal bovine serum. Preparation of hemin and SnPP Both hemin and SnPP were dissolved in 0. 2 N NaOH, adjusted to physiological pH 7. 4 with 1 N HCl, aliquoted in dark brown tubes and frozen at 80 C.
Astrocyte cultures Astrocytes were prepared from 16 to 22 week old aborted human fetal brain tissues obtained under a pro tocol approved Inhibitors,Modulators,Libraries by the Human Subjects Research Com mittee at our institution. Brain tissues were dissociated and resuspended in DMEM containing penicillin, streptomycin, gentamicin and Fungizone and plated onto poly L lysine coated 75 cm2 flasks at a density of 80 100106 cellsflask and incubated at 37 C in a 6% CO2 incubator. Culture medium was changed at a weekly interval. On day 21, flasks were shaken at 180 200 rpm for 16 h followed by trypsinization with 0. 25% trypsin in HBSS for 30 min. After adding FBS, centrifugation and washing, cells were seeded into new flasks with DMEM followed by medium change after 24 h. The subculture procedure was repeated four times at a weekly interval to achieve highly purified astrocyte cultures which were plated onto 60 mm petri dish, 6 or 12 well or 48 well plates CHIR99021 solubility for protein collection, RNA extraction or ELISA assay. Cell culture treatment conditions Astrocyte culture medium was replaced with DMEM without serum prior to SnPP or hemin treatment. The final serum concentration of 6% was restored at 3 h after the last hemin treatment unless noted.