Tris hcl buffer

Diazinon standard (> 98 %) from Supelco Company (USA), di-nitrophenyl-hydrazene (DNPH), acetonitrile (ACN, HPLC grade) from Caledon (Canada), solid phase extraction (SPE) cartridge octadecyl (C18) from Teknokroma (Spain), trichloro acetic acid (TCA) and TRIzol reagent from Roche (Swiss), tripure isolation reagent and expand reverse transcriptase from Roche Applied Sciences (Germany), tris-HCl buffer, FeCl₃, KCL, MgCl₂, NaH₂PO₄, EDTA, glucose, sucrose from Merck (Germany), technical DZN from Shimi-Keshavarz Pesticide Production Co. (Iran), primers from Gen Fanavaran Co. (Iran), and SYBR^® Premix Ex Taq from Takara Bio Inc. (South Korea) were used in this study.

C2C12 myoblasts were plated into 6-well plates (5 × 10⁴ cells/well) and grown until confluent. Incubation, with increasing concentrations of DEX (0.01–100 μM) and four different mixtures of R(+)LA and HMB (30 μM and 100 μM, 30 μM and 300 μM, 100 μM and 300 μM, 100 μM and 1000 μM, respectively), was allowed for 48 h. Concurrently, 1 μM DEX was incubated with increasing concentrations of R(+)LA (1, 10, 30, 100, 300 µM) and HMB (30, 100, 300, 1000, 3000 µM), either alone or in combination, for 48 h. After treatment, cells were scraped into 100 μL of lysis buffer (200 mM tris-hydrogen chloride (Tris-HCl) buffer, pH 7.5, containing 2 M NaCl, 20 mM ethylenediaminetetraacetic acid (EDTA), and 0.2% Triton X–100) (Merck, Milan, Italy). Fifty microlitres of the supernatant was incubated with 25 μM of the fluorogenic peptide caspase substrate, rhodamine 110 bis (N-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide) (Molecular Probes, Milan, Italy), at 25 °C for 30 min. The amount of cleaved substrate was measured in a 96-well plate fluorescence spectrometer (FlexStation 3, Molecular Devices; excitation at 496 nm and emission at 520 nm).

For HPLC analysis, chromatographic-grade methanol was used (VWR, Vienna, Austria). A Millipore purifier (Millipore, Burlington, MA, USA) was used to obtain water for HPLC. Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, phosphoric acid, potassium chloride, quercetin, hispiduline, ascorbic acid, ibuprofen, ketoprofen, sodium chloride, hydrogen peroxide, trypsin, Tris-HCl buffer, sodium nitroprusside, sodium salicylate, ferrous sulfate, sulfanilamide, naphthylethylenediamine dihydrochloride and perchloric acid were purchased from Sigma-Aldrich, Taufkirchen, Germany. Human albumin 20%—BB, 200 g/L was ordered from BB-NCIPD Ltd., Sofia, Bulgaria.

The sodium alginate (SA) was purchased from BDH Laboratory Supplies Poole, BH15 1TD, England. Folic acid (FA) 98% was purchased from Loba Chemie PVT.LTD-Mumbai, India. The Human FOLR2 Protein (FRs) > 90% (Mw = 26.5 KDa) and Human Serum Albumin Protein (HSA) > 95% (Mw = 67.4 KDa) were purchased from ACRO Biosystems, Newark, DE, USA. The polyethylene oxide (PEO) (Mw = 600,000 Da), bovine serum albumin (BSA) < 96% (agarose gel electrophoresis), human plasma, Tris-HCl buffer, potassium chloride, and potassium ferricyanide/potassium ferrocyanide were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). All the other reagents were of analytical grade and were used without further purification. All the aqueous solutions were filtered using Milli-Q purified ultrapure water with a resistivity of 18.2 MΩ cm⁻¹.