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2 protocols using aclonifen

1

HPLC-MS/MS Multianalyte Quantification

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Methanol HPLC Ultragradient Gold, formic acid 99% and ammonia solution 25% were purchased from Carlo Erba Reagents, SAS (Marseille, France). H2O - Pierce™. Water, acetonitrile and formic acid LC-MS grade Optima® were from Fisher Chemical (Hampton, NH, USA), state abbrev if USA, country). Caffeine-13C3 (99%, 1 mg/mL in methanol) was obtained from Sigma-Aldrich (Schnelldorf, Germany). Standards (purity ≥97%) of all the chemicals listed (tert-butylazine; desethyl-tert-butylazine; dimethoate; atrazine; desethylatrazine; tebuconazole; alachlor; omethoate; bentazone; diuron; linuron; chlorotoluron; cymoxanil; thiamethoxam; clothianidin; imidacloprid; acetamiprid; thiacloprid; simazine; isoproturon; cybutryne; methiocarb; quinoxyfen; chlorpyrifos; 2,4D; MCPP; aclonifen; bifenox; MCPA; dichlorvos; bezafibrate; ibuprofen; amoxicillin·3H2O; azithromycin; diclofenac; α-ethinylestradiol; β-estradiol; erythromycin; clarithromycin; trimethoprim; ciprofloxacin; enrofloxacin; sulfamethoxazole; bisoprolol; carbamazepine) were purchased from Sigma-Aldrich.
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2

HEK293 Cells Manipulation and Luciferase Reporting

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Human embryonic kidney HEK293 cells were from ATCC (American Type Culture Collection
[ATCC], Manassas, VA, USA) and cell culture reagents and materials were from Carlo Erba Reagents (Cornaredo, Milano, Italy) . The enzymes required for cloning procedures were from Fermentas (Vilnius, Lithuania). The kits for plasmid extraction, beetle luciferin potassium salt (D-luciferin), and BrightGlo substrate were from Promega (Madison, WI, USA). Tumor Necrosis Factor-α (TNF, purity higher than 95%), phorbol myristate acetate (PMA), poly(2-hydroxyethyl methacrylate) (poly-HEMA), gelatin from porcine skin type A, L-15 Medium (Leibovitz), isoproturon, naphthalene, aclonifen, hydrogen peroxide, and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Polydimethylsiloxane (PDMS) Silgard 184 pre-polymer and curing agent were from Dow Corning (Midland, MI).
The mammalian expression plasmids pGL4.32_NFkB_Luc2P and pGL4.74[hRluc/TK] carrying Luc2P luciferase under the regulation of NFkB transcriptional regulation and hRLuc under Herpes simplex virus thymidine kinase promoter (TK) were from Promega. Plasmids PpyGR-TS and red-emitting mutant PpyRE-TS luciferase genes were previously described (Branchini et al., 2007) . The reporter vectors pCMV_PpyGR-TS, pCMV_PpyRE-TS, pTK_PpyGR-TS, and pNFkB_PpyRE-TS were obtained by standard molecular cloning procedures.
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