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2012b). Briefly, aortic strips (two preweighed whole sections of aortae for each single measurement) were initially incubated with PSS at 37��C for 30 min, followed by a 10 min exposure to PSS, AA (30 ��m) or PGH2 (10 ��m) in 1000 ��l PSS (37��C). Thereafter, the reaction solution was pipetted into another tube, and the proportion of metabolites in tissues was further extracted with acetone, followed by drying with a stream of N2 and dissolving with the original reaction solution. Sample or standard solution (25 ng 6-keto-PGF1��, or 6-keto-PGF1��, TxB2, PGF2��, PGE2 and PGD2 at the indicated amounts; Cayman Chemical) further underwent solid-phase extraction as described elsewhere (Liu et al. 2012b). The HPLC system (Agilent 1100; Agilent www.selleckchem.com/products/GDC-0449.html Technologies, Santa Clara, CA, USA) was equipped with an HPLC column (Symmetry? C18, 3.5 ��m, 2.1 mm �� 100 mm; Waters, Milford, MA, USA). The mobile phase was methanol/10 mm ammonium acetate (50/50, pH 7.5) with a flow rate of 0.2 ml min?1. The amount of 6-keto-PGF1�� was calculated from the area of signal relative to that of a standard (25 ng), and was expressed in nanograms per milligram of wet tissues. The expression of COX-1, COX-2 or ��-actin in abdominal aortae was detected by real-time PCR. Preparation of RNA was performed using an RNAiso? Plus kit (TaKaRa, Dalian, China), Moroxydine according to the manufacturer's instructions. First strand cDNA was synthesized using total RNA (250 ng) and Oligo(dT)15 primers (TakaRa). The PCR primers for COX-1 were 5��-CCA ACT GTA CCA TCC CTG AG-3�� (sense) and 5��-AAT TCC CAG AGC CAG TAT CC-3�� (antisense); those for COX-2 and ��-actin were described previously (Liu et al. 2012b, 2013). Real-time PCR was performed using the SYBR? PrimScript? RT-PCR kit (TaKaRa). Data were expressed as means �� SEM from n numbers or pools of vessels from different animals. Student's unpaired t test (two tailed) was used to compare the difference between two means for statistical evaluation. When more Temozolomide ic50 than two means were compared, a one-way ANOVA followed by Dunnett's post hoc test or two-way ANOVA with Bonferroni's post hoc test was used. A value of P