Primary mouse anti human HO 1 or rabbit anti GFAP antibodies was

Primary mouse anti human HO 1 or rabbit anti GFAP antibodies was added and incubated overnight at 4 C. After washing, secondary antibody was added for 1 h at RT followed by selleck chemicals viewing under fluorescent microscope. pLEX HO 1 vector transfection Astrocyte cultures were transfected with 1 ug pLEX vector containing either blank or human Inhibitors,Modulators,Libraries HO 1 sequences under a cytomegalovirus promoter in Fugene 6 reagent for 72 h prior to IL 1b treatment for 72 h. Total cell pro teins were collected with M PER, aliquoted and mixed with 2 sample buffer before being stored at 20 C. Real time polymerase chain reaction Total RNA extracted from astroyctes Inhibitors,Modulators,Libraries after treatment Inhibitors,Modulators,Libraries was treated with DNase and reverse transcribed to cDNA with oligo 12 18, random hexmer, dNTPs, RNase inhibitor and SuperScript III reverse transcrip tase.

Mixtures of diluted cDNA, primers and SYBR Pre mix Ex Taq or SYBR Advantage qPCR premix were subjected to real time PCR according to manufactures protocol. Primer sequences were sense for HPRT. Western blot Cell lysates collected after treatment were electrophor ezed in 12% or 7. 5% acrylamidebis acrylamide, electrotransfered onto nitrocellulose membrane and probed Inhibitors,Modulators,Libraries with antibodies for HO 1, HO 2, iNOS or MAPKs fol lowed by alkaline phosphatase conjugated secondary antibodies with chemiluminescence detection using Kodak Image Station, New Heaven, CT. Statistical analysis Data are expressed as meanSD or SE as indicated. For comparison of means of multiple groups, analysis of var iance was used, followed by Fishers PLSD test.

Results Inhibition of iNOS mRNA expression and NO production To test the hypothesis that hemin would inhibit iNOS expression, human astrocyte cultures were pretreated with hemin for 24 h followed by IL 1b treatment for 4 h or 24 h for total RNA isolation. Inhibitors,Modulators,Libraries Marked inhibition of iNOS mRNA expression was observed in hemin pre treated cells. In the same hemin treatment paradigm followed by stimulation of astrocytes with IL 1b for 72 h, a similar inhibitory effect of hemin was observed when culture supernatants were assayed for NO. Hemin induced HO 1 expression in human astroyctes To confirm that hemin induces HO 1 in human astro cytes, cells were treated with hemin for 24, 48 and 72 h and HO 1, HO 2 and b actin expression were assessed by western blot. Induction of HO 1 expression by hemin was robust at 24 h and decreased over time, while HO 2 was constitu tively expressed in human astrocytes. No effect of hemin on b actin expression was observed. There was no cyto toxicity induced by hemin measured by MTT or alamarBlue assays. Immunocytochemical reaction also demonstrated no nuclear fragmentation in hemin Calcitriol IL-2 treated astrocytes indicating no cytotoxicity.

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