The culture medium was refreshed the next day to get rid of debris. The neuronal purity as determined by Microtubule associated protein 2 staining was around www.selleckchem.com/products/Y-27632.html 98%. Cultures were used after 5 days in vitro. Induction of excitotoxicity Cortical neuron cultures were subjected Inhibitors,Modulators,Libraries to an excito toxic challenge with glutamate, after which cultures were refreshed with fresh media and were incubated at 37 C. Supernatants from neuron cul tures were collected 18 hours after glutamate challenge and were applied to the primary astrocyte cultures. Primary astrocyte cultures Primary astrocyte cultures were established from cere bral cortices of postnatal C57BL 6 J and A2B KO mice according to a previously described pro cedure, which was modified to reduce microglial contamination.

Microglial cells were separated from the astrocytic monolayer by 1 hour shake off at 150 rpm. This procedure was repeated two times with an interval Inhibitors,Modulators,Libraries of 4 days in vitro between each shake off, fol lowed by an overnight shake off at 240 rpm to remove oligodendrocyte precursor cells. Purified astrocytes were washed Inhibitors,Modulators,Libraries with HBSS buffer containing 1 mM ethylenedia minetetraacetic acid and further detached using HBSS with 0. 1% trypsin. Cells were reseeded with fresh astrocyte culture medium in multi well plates and maintained in culture to con fluency. To further reduce microglial contamination, confluent astrocyte cultures were treated with 5 mM LME, a lysosomotropic Inhibitors,Modulators,Libraries agent, for 4 to 5 hours. Astrocytes were ready for experiments after 1 to 2 days. Our cell preparations had a high percentage of astro cytes, which was confirmed by immunostaining against GFAP and CD11b.

Real time polymerase chain reaction Total RNA of primary astrocytes was extracted, Inhibitors,Modulators,Libraries puri fied and transcribed into cDNA as described previ ously. Quality of the cDNA was examined using the following housekeeping gene Glyceraldehyde 3 phosphate www.selleckchem.com/products/3-deazaneplanocin-a-dznep.html dehydrogenase primer pairs, Fw. The effect of neuronal supernatants and NECA on LIF mRNA expression in cultured astro cytes was analyzed by real time PCR using the iCycler and iQ SYBR Green supermix. Mouse Hypoxanthine phos phoribosyltransferase 1 and GAPDH primers were used for normalization to housekeeping genes, and these genes showed no variations in response to the experimen tal treatments. The primer pairs used for qPCR were, LIF and Accession number, NM 013556. The comparative Ct method, was calculated by, and was used to determine the relative gene expression levels. Western blot Western blotting on cultured cortical astrocytes was per formed as previously described. Equal amounts of protein were loaded to 12. 5 or 15% sodium dodecyl sulfate polyacrylamide gels and subsequently transferred to PVDF membranes.