-3′ 5′-ACATTCGAGGCTCCAGTGAATTCGG-3′ 5′-GAGGATACCACTCCCAACAGA-3′ 5′-AAGTGCATCATCGTTGTTCATACA-3′ 5′-TGAAGGAGGCCACCAAGGAGG-3′ 5′-AGAGGTCACCCAGGTAGCGGG-3’Figure five. The effect of AM-EO around the mRNA expression levels of iNOS, COX-2, TNF-, IL-6 and HO-1 in LPS-stimulated RAW 264.7 macrophages.Int. J. Mol. Sci. 2013,Figure six. The quantitative mRNA ratio of (A) iNOS, (B) COX-2, (C) TNF-, (D) IL-6 and (E) HO-1. Each value represents the mean ?SD (n = three). Groups sharing the same superscript letter are usually not considerably unique (p 0.05) as revealed by Dunnett’s post hoc tests.3. Experimental Section three.1. Essential Oil and Cell Line Steam-distilled important oil of Achillea millefolium L. (AM-EO) was bought from Australian Botanical Merchandise, Pty Ltd.2749963-99-1 manufacturer (Hallam, Victoria, Australia). The murine macrophage cell line RAW 264.7 (BCRC 60001) was obtained in the Bioresource Collection and Investigation Center (BCRC, Hsinchu, Taiwan) and was utilised in anti-inflammatory activity assays. 3.two. Materials Fetal bovine serum (FBS), L-glutamine, penicillin-streptomycin, trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA), deoxynucleotide triphosphate (dNTP), oligo(dT), Taq DNA polymerase and Dulbecco’s Modified Eagle Medium (DMEM) medium had been bought from Gibco BRL/Invitrogen (Carlsbad, CA, USA). Lipopolysaccharide (LPS; from Escherichia coli, serotype O111: B4), 3-(four,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Griess reagent, sodium nitrite, pyrogallol, nitro blue tetrazolium (NBT), 2-nitrobenzoic acid, nicotinamide adenine dinucleotide phosphate (NADPH), hydrogen peroxide option (H2O2), bovine serum albumin (BSA), ethidium bromide, dithiothreitol (DTT), agarose along with other chemical compounds were purchased from Sigma-Aldrich (St. Louis, MO, USA). Deionized distilled water (ddH2O) applied to prepare solutions and buffers had been purified using a Milli-Q program (Millipore, Bedford, MA, USA).Int. J. Mol. Sci. 2013, 14 3.3. Gas Chromatography and Mass Spectrometry AnalysisGC-MS analyses had been carried out on a GCMS-QP-2010 plus Gas chromatograph Mass Spectrometer (Shimadzu, Japan) and utilizing GCMS-solution software (v. 2.50 SU3, Shimadzu, Japan). Compounds had been separated on a Forte ID-BPX5 cross-linked 5 phenyl-95 methyl polysiloxane (30 m ?0.25 mm internal diameter (i.d.), film thickness 0.25 m) capillary column (SGE Analytical Science, Ringwood, Victoria, Australia). The column was maintained at 50 for 5 min right after injection, then programmed at five /min to 150 , and ultimately, programmed at 10 /min to 300 .2-(Tributylstannyl)thiophene site The injection volume comprised 1.0 of pure necessary oil with a split ratio of 1:one hundred. Helium was utilised because the carrier gas at a continual flow-rate of 1.PMID:23962101 0 mL/min. The injector, transfer line and ion-source temperatures had been 250, 230 and 250 , respectively. MS detection was performed with an electron impact mode at 70 eV ionization energy and 60 A ionization existing, operating in the full-scan acquisition mode within the 40?50 amu range. Compounds had been identified by comparing the retention instances and retention indices in the chromatographic peaks using a regular library, National Institute of Requirements and Technologies (NIST) MS spectral database (version 2005, NIST, Gaithersburg, MD, USA, 2005) and comparing the measured Kovats index (KI) to a homologous series of n-alkanes (C5 26). 3.4. Cell Culture RAW 264.7 cells had been cultured in DMEM supplemented with 10 FBS, two mM L-glutamine and 1 penicillin-streptomycin (one hundred U/mL penicillin and 100 g/mL streptomycin).