Selected article for: "Quantity software and room temperature"
Author: Steelman, Andrew J; Li, Jianrong
Title: Poly(I:C) promotes TNFa/TNFR1-dependent oligodendrocyte death in mixed glial cultures
  • Document date: 2011_8_3
    • ID: 16032h3d_15
    Snippet: Transcriptional expression of Iba-1, GFAP, TLR3, TNFα, IL-1β, and IL-6 in microglia and astrocyte monocultures upon stimulation with poly(I:C) was examined as described previously [25] . Briefly, cells were plated onto poly-D-lysine coated 6-well plates at a density of 1.0 × 10 6 cells per well in DMEM containing 10% fetal bovine serum. After 24 h, cells were washed twice with BDM medium, and then treated with poly(I:C) (50 μg/ ml) or vehicle.....
    Document: Transcriptional expression of Iba-1, GFAP, TLR3, TNFα, IL-1β, and IL-6 in microglia and astrocyte monocultures upon stimulation with poly(I:C) was examined as described previously [25] . Briefly, cells were plated onto poly-D-lysine coated 6-well plates at a density of 1.0 × 10 6 cells per well in DMEM containing 10% fetal bovine serum. After 24 h, cells were washed twice with BDM medium, and then treated with poly(I:C) (50 μg/ ml) or vehicle control (PBS) for 0-24 h. RNeasy kits were used to extract RNA according to the manufacturers' instructions (Qiagen, Valencia, CA). Residual DNA was digested by incubating RNA samples with DNase I for 15 min at room temperature followed by DNase inactivation at 65°C for 10 min according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). The samples were reverse transcribed to cDNA using the reverse transcription system kit (Promega, Madison, WI) as described previously [25] . The purity of the mono-cultures was determined using specific primers for GFAP (astrocyte marker) and Iba-1 (microglia marker). Primers specific for β-actin were used as a loading control. All products were amplified by PCR using 100 ng of cDNA and the following primers: TLR3, forward-TGCGATTGGCAAGTTATTCG, reverse-GCGGAG GCTGTTGTAGGAAA; TNF, forward-GCCCACGTCG-TAGCAAAC, reverse-GCAGCCTTGTCCCTTGAA; IL-1β, forward-TGACCCATGTGAGCTGAAAG, reverse-AGGGATTTTGTCGTTGCTTG; IL-6, forward-CAG-GAACGAAAGTCAACTCCA, reverse-ATCAGTCC-CAAGAAGGCAACT; CCL2, forward-CCAGCCCA GAAACCAGCCAACTC, reverse-GCATCTGGCTGA-GACAGCACGT; CCL5, forward-AGCAGCAAGTGC TCCAACCTTG, reverse-GCACACCTCCCAGGCCA-TAGGA; GFAP, forward-CAGCTT CGAGCCAAG-GAG, reverse-TGTCCCTCTCCACCTCCA; Iba1, forward-CTTTTGGACTGCTGAAAGCC, reverse-GTT TCTCCAGCATTCGCTTC; and β-actin, forward-AGACTTCGAGCAGGAGATGG, reverse-CCATCAT-GAAGTGTGACGTTG. Products were electrophorezed on 2% agarose gels and visualized under UV light using a Bio-Rad Chemidoc XRS gel documentation system and Quantity-one software. Quantitative PCR was used to determine fold increase in CCL2 and CCL5 relative to β-actin using the formula 2 -ΔΔCt as described previously [30] .

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