Sulphorhodamine b srb

Polyethylene imine (PEI), Polylactide-co-glycolide (PLGA), Polylactic acid (PLA), Alginate (Low viscosity, 2%), Platinum octaethyl porphyrin (Pt-Porphyrin), o-dianisidine, Horseradish Peroxidase (HRP), Glucose, Glucose Oxidase (Type VII obtained from Aspergillus Niger) (GOx), Poly-vinyl alcohol (PVA) and Glucose were procured from Sigma Aldrich, India. Calcium chloride, Dicholoromethane, Acetone, Ethanol, Tween 85, Dimethyl sulphoxide (DMSO) and Sodium lauryl suphate (SLS) were procured from Merck Mumbai, India. All chemicals were reagent grade and were used as received. L929 (Mouse fibroblast) cell line was procured from National Centre for Cell Science (NCCS), Pune, India. DMEM (Dulbecco’s Medium Eagle Medium, Sigma, USA), FBS (Fetal Bovine Serum, Sigma, USA), trypsin-EDTA solution, trichloroacetic acid (TCA, Loba Chemie, India) and Sulphorhodamine B (SRB), Sigma-Aldrich Chemie, USA) were procurd to use them for cell culture experiments.

Sulphorhodamine B (SRB) (Sigma, St. Louis, MO, USA) dye assay was performed to determine cell viability, as previously described [54 (link)]. Briefly, gastric cancer cell lines were seeded at the density of 3 × 10³ cells/well in 96-well plates. After 24 h of culture, cells were treated with different doses of RSV (ranging from 5 up to 200 μM) for 72 h. Then, cells were fixed, washed, and incubated with 50 µL of SRB solution (0.1% weight/volume in 1% acetic acid) per well and incubated at room temperature for an additional 30 min. Unbound SRB was removed by washing with 1% acetic acid. Plates were air-dried, and the retained SRB was solubilized with 100 µL of 10 mM Tris base (pH 10.5). Optical densities were read in a spectrophotometer plate reader at 540 nm. Values are shown as mean ± S.D., n = four independent experiments in triplicate.

DNA topoisomerase I (Topo I, human) and sulphorhodamine B (SRB) were acquired from Sigma-Aldrich (St Louis, MO, USA). Amicon Ultra-0.5 centrifugal filters (3 kDa) and ultrapure water were obtained from Millipore Co. Ltd. (Bedford, MA, USA). Methanol and Acetonitrile (HPLC grade) were purchased from Merck (Darmstadt, Germany). Other chemicals were analytical grade and supplied by Aladdin industrial corporation (Shanghai, China). Fetal bovine serum (FBS), plasmid pBR322, 4 S Green Plus nucleic acid dye and TAE buffer were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). A549 cells were supplied by Beyotime Institute of Biotechnology (Haimen, China). DNA topoisomerase I (Topo I, human) was acquired from Sigma-Aldrich (St Louis, MO, USA).

Folin-Ciocalteu reagent, aluminum chloride, chlorogenic acid, quercetin, bergapten, fetal bovine serum (FBS), L-glutamine, penicillin/streptomycin, Dulbecco’s Modified Eagle’s Medium (DMEM), bovine serum albumin (BSA), protease inhibitors, trypan blue, phosphate buffered saline (PBS), LPS, trichloroacetic acid (TCA), sulphorhodamine B (SRB) were purchased by Sigma-Aldrich S.p.a. (Milan, Italy). ECL System was from Bio-Rad. RAW 264.7 cells were purchased by ATCC, UK (No. TIB-71). The Abs employed were anti-phosphoJak2 (#PA538287), anti-Jak2 (#PA511267), anti-phosphoStat3 (#PA5121259), anti-Stat3(#PA5120138), (all from ThermoFisher Scientific, Waltham, MA, USA), anti-β-actin (AC-15; sc-69879) from Santa Cruz Biotechnology, Inc., Heidelberg, Germany. All other reagents were obtained by VWR International s.r.l. (Milan, Italy).

Curcumin (CUR, ≥94% curcuminoid content, Sigma-Aldrich, St. Louis, MO, USA), methotrexate (MTX, >99% pure, Fermion, Espoo, Finland), poly(ε-caprolactone) (PCL, Mw 10,000–14,000 g.mol⁻¹, Sigma-Aldrich), poly(ethylene glycol) 6000 (PEG, Mw 5,400–6,600 g.mol⁻¹, Cromato Produtos Químicos, Diadema, Brazil), sorbitan monooleate (Span 80, Oxiteno, Mauá, Brazil), polysorbate 80 (Tween 80, Delaware, Porto Alegre, Brazil), medium chain triglycerides (MCT, 99% pure, Focus Química, São Paulo, Brazil), acridine orange base (AO, Sigma-Aldrich), ethidium bromide (EB, Sigma-Aldrich), methylthiazolyldiphenyltetrazolium bromide (MTT, Sigma-Aldrich), sulpho-rhodamine B (SRB, Sigma-Aldrich), penicillin-streptomycin (Sigma-Aldrich), and acetone (≥99.9% pure, Vetec Química, Rio de Janeiro, Brazil) were used as received. HPLC-grade methanol was purchased from Tedia (Rio de Janeiro, Brazil). RPMI 1640 medium and fetal bovine serum were obtained from Vitrocell (Campinas, Brazil). Water was purified in a Milli-Q Plus water purification system (Millipore, Bedford, MA, USA). All other solvents and reagents were analytical grade. The Calu-3 cell line was obtained from the Bank of Cells of Rio de Janeiro (BCRJ, Brazil) and was kindly provided by Dr. Katia Sabrina Paludo.

All the glassware was washed with aqua regia [HCl:HNO₃ = 3:1 (v/v)] and then rinsed with deionized water. Tetrachlorauric acid (HAuCl₄·3H₂O), 6-mercaptopurine, chitosan low molecular weight (mol wt 50,000–190,000 Da), dimethylsulphoxide (DMSO), RPMI-1640 medium, sodium bicarbonate, trypan blue, fetal Bovine Serum (FBS), trypsin, acetic acid, sulphorhodamine-B (SRB), trichloroacetic acid (TCA) and tris base 10 mM (PH 10.5) were obtained from Sigma Aldrich Chemical Co., St. Louis, Mo, USA.
The human breast carcinoma cell line (MCF7) was obtained from the American Type Culture Collection (ATCC, MO, USA). The tumor cell line was propagated and maintained by serial sub-culturing in RPMI-1640 medium containing 10% FBS and 1% penicillin/streptomycin.