Hplc grade h2o

FCS in DMEM was reduced to 0.1%, dependent on the following experiments, 16 h prior incubation. FCS was reduced to elucidate the potential effect of MTXs on cholesterol or other lipids being also present in FCS. MTXs were incubated with a concentration of 100 µM for 24 h (8 + 16 h), whereas controls were treated with HPLC-grade H₂O (Fisher Scientific, Schwerte, Germany) as a solvent control. For experiments with γ-secretase inhibitor IX, cells were pretreated 2 h with 2.5 µM γ-secretase inhibitor, before incubation with MTXs was performed as described above. For determination of cholesterol level, cells were long-term incubated for six days in DMEM reduced to 1% FCS.

Analytical-scale HPLC was performed with an Agilent 1200 series system on octadecyl carbon chain (C18)–bonded silica columns (5-μm pore size, 4.6 × 250 mm, 218TP54, Vydac). The solvent system was HPLC-grade H₂O (W/0106/PB17, Fisher Scientific) and HPLC-grade acetonitrile (ACN; A/0627/PB17, Fisher Scientific). Buffer A consisted of HPLC-grade H₂O containing 0.1% (v/v) HPLC-grade TFA (T/3258/04, Fisher Scientific), and buffer B consisted of 80% (v/v) ACN in HPLC-grade H₂O containing 0.1% (v/v) TFA.
Mass spectrometry analysis was performed on the Agilent Series 1100 LC-MS system in positive mode of ESI. The solvent system was as follows. Buffer A consisted of LC-MS grade H₂O (W/0112/17, Fisher Scientific) containing 0.1% (v/v) formic acid (06440, Fluka), and buffer B consisted of 80% (v/v) ACN in MS-LC–grade H₂O containing 0.1% (v/v) formic acid. Data analysis was performed with LC/MSD ChemStation software.
SEC-MALS was performed on a Superdex 200 5/150 GL column (GE Healthcare) in SEC-MALS buffer (50 mmol/liter Tris-HCl (pH 7.4), 5 mm MgCl₂, 1 mmol/liter CaCl₂, 100 mm NaCl, and 100 mmol/liter tris(2-carboxyethyl)phosphine). Recombinant troponins were extensively dialyzed against SEC-MALS buffer before experiments. Light scattering and refractive index were measured on a Mini DAWN and OPTILAB DSP (Wyatt Technology, UK), respectively. Data were analyzed with custom-written software.

Glacial acetic acid, Folin-Ciocalteu phenol reagent, 2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt (ABTS), potassium persulfate, ferric chloride, sodium acetate, 2,4,6-tripyridyl-s-triazine (TPTZ), rutin, isoflavone aglycones, including daidzein, genistein, and glycitein, were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), and three isoflavone glycosides, including genistin, daidzin, and glycitin, were purchased from Indofine (Hillsborough, FL, USA). HPLC-grade H₂O, methanol, and acetonitrile were purchased from Fisher Scientific (Fairlawn, OH, USA). All chemicals were of analytical grade.

Kanamycin sulfate, isopropyl β-D-1-thiogalactopyranoside (IPTG), 100X MEM vitamin mixture (for bacterial culture work), bovine serum albumin (BSA), Bromophenol Blue, 5-bromouracil (BrU), 5-bromouridine (BrUrd), 3-(trimethylsilyl)-1-propanesulfonic acid-d₆ sodium salt (DSS-D₆), 5-fluorouracil (FU), 5-fluorouridine (FUrd), nitroblue tetrazolium (NBT), riboflavin, uracil (U) and uridine (Urd) were purchased from Sigma (Oakville, Canada). 5-chlorouracil (ClU) was obtained from Ark Pharm Incorporated (Arlington Heights, USA). The trifluridine (F3TDR) was purchased from Oxchem Corporations (Wood Dale, USA). The BLUelf prestained protein ladder was purchased from FroggaBio (Concord, Canada). The Luria Broth (LB, Invitrogen), Coomassie G-250, HPLC grade H₂O and the Spectra/Por 6–8 kDa molecular weight cut-off (MWCO) dialysis tubing were purchased from Fisher Scientific (Ottawa, Canada). The 3.5 kDa MWCO Snakeskin dialysis tubing was purchased from Thermo Fisher Scientific (Whitby, Canada). The ¹⁵NH₄Cl was purchased from Cambridge Isotope Laboratories (Andover, USA). The MAXYMum Recovery 1.5 mL centrifuge tubes were purchased from Corning (Fairport, USA). The 3 mM SampleJet NMR tubes were obtained from Bruker (Milton, Canada).