05 mg ml gentamycin, and seeded in tissue culture flasks After <

05 mg ml gentamycin, and seeded in tissue culture flasks. After first 48 hr culturing at 37 C and 5% CO2, the cells were washed and cultured with Inhibitors,Modulators,Libraries 2% FBS for 4 to 5 days. The flasks were then shaken and microglia were harvested, washed and plated on sub strates and at densities appropriate for each assay. Chemicals Inhibitors,Modulators,Libraries Classical activation was evoked using 10 ng ml LPS from E. coli K 235, as before. Alternative activation was evoked with 20 ng ml recombinant rat IL4, as before. For the transmigration and invasion assays, microglia were treated 1 hr after either stimulus with one of the following inhibitors. The broad spectrum MMP inhibitor, GM6001 has Ki values from 0. 2 to 27 nM depending on the MMP, and the heparanase inhibitor, OGT 2115 has an IC50 of 0. 4 uM.

The cysteine protease inhibitor, E 64, was used to inhibit cysteine cathepsins. The select ive Cat S inhibitor has a Ki value of 185 pM, and the selective Cat K inhibitor Inhibitors,Modulators,Libraries I 2 propanone has a Ki of 22 nM. All inhibitory constants were according to the suppliers. Stock solutions were made in DMSO, sterile double distilled water or sterile phosphate buffered saline with 0. 3% bovine serum albumin. For all inhibitors, aliquots were stored at ?20 C. ATP was prepared just before use. Quantitative real time reverse transcriptase polymerase chain reaction To monitor gene transcript levels, 500,000 cells were seeded into each 35 mm culture dish, and our standard protocol was used, as recently described. Gene specific primers were designed using Primer3Output.

After 24 hr treatment with LPS or IL4, total RNA was extracted from primary microglia using the TRIzol method, followed by RNeasy Mini Kit for further Inhibitors,Modulators,Libraries purifi cation. A two step reaction was performed according to the manufacturers instructions. In brief, total RNA was reverse transcribed in 20 ul volume using 200 U of SuperScriptII RNase reverse transcriptase, with 0. 5 mM dNTPs and 0. 5 uM oligo dT. Amplification was performed on an ABI PRISM 7700 Sequence Detection System at 95 C for 10 min, 40 cycles at 95 C for 15 s, and 56 C for 20 s. No tem plate and no amplification controls were included for each gene, and melt curves showed a single peak, confirming specific amplification. The threshold cycle for Inhibitors,Modulators,Libraries each gene was determined, and normalized to that of the housekeeping gene, hypoxanthine guanine phosphoribosyl transferase, which we find to be especially stable in primary rat microglia under all treatments we have investigated.

Results are expressed as relative mRNA expression from four separate microglia cultures grown from four different rat pups. Immunocytochemical analysis The methods were similar to our recent paper. Microglia were seeded at 60,000 cells per UV irradiated 15 mm glass coverslip. They were cultured for 1 day in MEM with 2% MG132 DMSO FBS, and then fixed in 4% paraformaldehyde at room temperature for 15 min. Cells were permeabilized with 0.

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