All solvents used for HPLC analysis were of liquid chromatography grade and the water was ultrapure. From Sigma-Aldrich (St. Louis, MO, USA), the following products were obtained: Folin–Ciocalteu reagent, α-tocopherol, γ-tocopherol, trolox, butylated hydroxytoluene, 1,1,3,3-tetraethoxy propane and cinnamtannin A2, while acetone, butanol, isopropanol, hexane, acetonitrile and methanol were obtained from Panreac (Castellar del Vallés, Barcelona, Spain). Finally, the following standard compounds were purchased at Extrasynthese (Genay, France): gallic acid, catechin, epicatechin, epicatechin gallate, and procyanidins B1, B2, B3 and C1.
Isopropanol
Manufactured by ITW Reagents
Sourced in Spain
Isopropanol is a clear, colorless, volatile, flammable, and water-miscible liquid. It is commonly used as a solvent and disinfectant in various industrial and laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Acetone, by ITW Reagents (5 mentions)
Acetonitrile, by ITW Reagents (3 mentions)
Hexane, by ITW Reagents (2 mentions)
Butanol, by ITW Reagents (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Methanol, by ITW Reagents (2 mentions)
Ethanol, by ITW Reagents (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Epicatechin, by Extrasynthese (1 mentions)
Butylated hydroxytoluene, by ITW Reagents (1 mentions)
Catechin, by Extrasynthese (1 mentions)
Epicatechin gallate, by Extrasynthese (1 mentions)
Gallic acid, by Extrasynthese (1 mentions)
Folin ciocalteu reagent, by ITW Reagents (1 mentions)
Trolox, by ITW Reagents (1 mentions)
Anhydrous sodium sulfate, by ITW Reagents (1 mentions)
Oxalic acid, by ITW Reagents (1 mentions)
N hexane, by ITW Reagents (1 mentions)
Zirconium tetrachloride zrcl4, by Thermo Fisher Scientific (1 mentions)
Hydrochloric acid (hcl), by ITW Reagents (1 mentions)
12 protocols using isopropanol
1
HPLC Analysis of Antioxidant Compounds
2
Fatty Acid Methyl Ester Analysis
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Fatty acid methyl esters (Supelco 35 Component FAME Mix) were obtained from Sigma-Aldrich SL (Madrid, Spain). N-hexane, isopropanol, anhydrous sodium sulfate, undecanoic acid (C11:0), methyl acetate, sodium methoxide, methanol, oxalic acid, and diethyl ether were acquired from Panreac Química SL (Panreac AppliChem, Barcelona, Spain) and Sigma-Aldrich (Merck, Munich, Germany). All reagents were of analytical grade.
3
Antitumor Potential of Algae Extracts
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The antitumor capacity of algae extracts on cell viability and cell proliferation studies were measured using MTT method. This assay is based on cleavage of the tetrazolium salt MTT by mitochondrial dehydrogenases of viable cells to form a blue formazan product. The formazan produced is proportional to the number of living cells present and can be measured colorimetrically (Yuan and Walsh 2006 (link)). After the treatment with the methanol and dichloromethane extracts, the cells medium was removed and cells were washed with Hanks’ medium (medium composition in mM: NaCl 137, KCl 5, MgSO4 0.8, Na2HPO4 0.33, KH2PO4 0.44, CaCl2 0.25; MgCl2 1.0, Tris HCl 0.15 and sodium butyrate 1.0, pH 7.4). After that, cells were incubated with MTT (1.2 mM) (Sigma, Germany), previously dissolved in Hanks’ medium, during 4 h at 37 °C. The formazan products were dissolved in isopropanol (Panreac, Spain) with 0.04 N HCl and the absorbance (Abs) was read at 570 nm (Biotec, Synergy H1 hybrid reader).
4
Fabrication of SERS-active Au NPs/pZrO2 Substrates
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Polystyrene nanoparticles (PS NPs) of diameters (DP = 150, 250, and 350 nm) were deposited onto cleaned Si substrates and were slightly annealed at 50 °C for 10 min before spin-coating with a precursor solution of ZrO2. The ZrO2 precursor was prepared with a mixture of zirconium tetrachloride (ZrCl4, 98%, Acros Organics, Pittsburgh, PA, USA) and isopropanol (99.8%, Panreac Applichem, Darmstadt, Germany) that was left to stand for ∼24 h until it achieved a gel-like consistency before use for spin-coating. ZrO2 was then allowed to form on the surface of the coated samples through annealing at 600 °C for 3 h to remove the template layer of PS NPs, leaving behind a regularly arranged concave porous ZrO2. With the use of an electron beam evaporator, Au NPs were deposited onto the porous ZrO2 substrate to produce SERS-active Au NPs/pZrO2 substrates. As Au NPs/pZrO2 substrates were fabricated at three varying PS NP sizes, resulting to a variation in the depth, pore, and pore wall sizes, these are denoted by Au NPs/pZrO2 (DP).
5
Cytotoxicity Evaluation using MTT Assay
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Cytotoxicity and cell proliferation tests were evaluated through the 3-4 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) method. This assay is based on cleavage of tetrazolium salt MTT by mitochondrial dehydrogenase of viable cells to form a blue formazan product. The formazan produced is proportional to the number of living cells present and can be measured colorimetrically.[22 (link)] After the treatment with the methanolic and dichloromethane extracts, the medium was removed and cells were washed with Hank's buffer (composition, in mM: NaCl 137, KCl 5, MgSO4 0.8, Na2 HPO4 0.33, KH2 PO4 0.44, CaCl2 0.25; MgCl2 1.0, Tris HCl 0.15, and sodium butyrate 1.0, pH = 7.4). Cells were then incubated 4 h at 37°C with 1.2 mM MTT (Sigma-Aldrich, Germany) which was previously dissolved in Hank's buffer. The formazan products were dissolved in isopropanol (Panreac, Spain) containing 0.04 N HCl and quantified spectrophotometrically at 570 nm.
6
In Vitro Cell Analysis Reagents
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Tyrosine ( purity: ≥98%), tyrosol (≥98%), hydroxytyrosol (≥98%), serotonin (≥98%) and protocatechuic acid (≥97%), fetal bovine serum (FBS), Rosewell Park Memorial Institute (RPMI) 1640 medium, non-essential amino acids (NEAA), minimal essential medium (MEM) vitamins, sodium pyruvate, L-glutamine, antibiotic-antimycotic solution, paraformaldehyde (PFA), bovine serum albumin (BSA) and universal mounting medium (DPX), Ringer HEPES solutions and sodium fluorescein were purchased from Sigma Aldrich (St Louis, MO, USA). Thiazolyl blue tetrazolium bromide (MTT) was obtained from Alfa Aesar (Haverhill, MA, USA). Dimethyl sulfoxide (DMSO), isopropanol, hydrochloric acid, and acetonitrile (ACN) were acquired from PanReac AppliChem (Barcelona, Spain). NuSerum IV was provided by Corning Costar Corp (New York, NY, USA). Triton X-100 was purchased from VWR International (Radnor, PA, USA). β-Catenin (rabbit, #71-2700), Alexa Fluor® 555 (goat anti-rabbit, #A21428), and Hoechst 33342 were acquired from Thermo Fisher Scientific (Waltham, MA, USA).
7
Production and Characterization of Bacterial Cellulose
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The bacteria strain K. xylinus (NCIMB 5346) was purchased from CECT (Spain). The culture media
was prepared using glucose, peptone, yeast extract purchased from
Condalab (Spain), Na2HPO4·12H2O, and citrate acid monohydrate from Sigma-Aldrich (Merck, Spain).
Human bone marrow-derived mesenchymal stromal cells (hBM-MSCs)
and human lung fibroblasts (HLFs) were purchased from ATCC (PCS-500-012)
as well as the culture media containing 125 pg/mL recombinant human
fibroblast growth factor (rh FGF), 15 ng/mL recombinant human insulin-like
growth factor-1, 7% fetal bovine serum (FBS), and 2.4 mMl -alanyl-l -glutamine. Human lung fibroblasts (HLFs) were
purchased from ATCC (PCS-201-013) as well as the culture media containing
7.5 mMl -glutamine, 1 μg/mL hydrocortisone hemisuccinate,
50 μg/mL ascorbic acid, 5 ng/mL rh FGF, 5 μg/mL recombinant
human insulin, and 2% FBS.
For all of the experiments, sodium
hydroxide (NaOH), acid acetic,
and paraformaldehyde (PFA) were purchased from Sigma-Aldrich, acetone,
and isopropanol from PanReac AppliChem (Spain), and phosphate-buffered
saline (PBS) 1× and 10×, Trypsin, and FBS from Gibco (Fisher
Scientific, Spain).
was prepared using glucose, peptone, yeast extract purchased from
Condalab (Spain), Na2HPO4·12H2O, and citrate acid monohydrate from Sigma-Aldrich (Merck, Spain).
Human bone marrow-derived mesenchymal stromal cells (hBM-MSCs)
and human lung fibroblasts (HLFs) were purchased from ATCC (PCS-500-012)
as well as the culture media containing 125 pg/mL recombinant human
fibroblast growth factor (rh FGF), 15 ng/mL recombinant human insulin-like
growth factor-1, 7% fetal bovine serum (FBS), and 2.4 mM
purchased from ATCC (PCS-201-013) as well as the culture media containing
7.5 mM
50 μg/mL ascorbic acid, 5 ng/mL rh FGF, 5 μg/mL recombinant
human insulin, and 2% FBS.
For all of the experiments, sodium
hydroxide (NaOH), acid acetic,
and paraformaldehyde (PFA) were purchased from Sigma-Aldrich, acetone,
and isopropanol from PanReac AppliChem (Spain), and phosphate-buffered
saline (PBS) 1× and 10×, Trypsin, and FBS from Gibco (Fisher
Scientific, Spain).
8
Enzymatic Synthesis of Silica Nanoparticles
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The reagents Tetraethoxysilane 98% (TEOS), n-dodecylamine 98%, buffer solution of enzyme Candida antarctica lipase B 5000 LU/g (CalB), titanium dioxide 99.5%, p-nitrophenyl butirate 98% (pNPB), valeric acid 99% and Bradford reagent were purchased from Sigma Aldrich. Ethanol 98% (EtOH), methanol 99.5% (MeOH), isopropanol 99.5% (iPrOH), 1-butanol 99.5% (BuOH), acetonitrile 99.9% and Cobalt (II) chloride hexahydrates 98% were purchased from Panreac and used without further purification.
9
Oleic Acid and Montmorillonite Clay Synthesis
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Fatty acid oleic acid (cis-9-octadecenoic acid, C18:1; 99%) and the commercial
clay (Montmorillonite KSF clay) were purchased from Fluka (Sigma-Aldrich,
Steinheim, Germany). Glycerol (1,2,3-propanetriol, ≥99.5%)
and isopropanol were purchased from Panreac (Barcelona, Spain). All
these chemicals were used as received without any further purification.
Acetonitrile (ACN), methanol (MeOH), and tetrahydrofuran (THF) were
all from Fisher and were of HPLC grade.
clay (Montmorillonite KSF clay) were purchased from Fluka (Sigma-Aldrich,
Steinheim, Germany). Glycerol (1,2,3-propanetriol, ≥99.5%)
and isopropanol were purchased from Panreac (Barcelona, Spain). All
these chemicals were used as received without any further purification.
Acetonitrile (ACN), methanol (MeOH), and tetrahydrofuran (THF) were
all from Fisher and were of HPLC grade.
10
Chemical Reagents for Bioassays
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Nitric acid, ethanol, acetone, isopropanol, sodium carbonate decahydrate and sodium phosphate monohydrate were purchased from Panreac (Barcelona, Spain). Light petroleum, pyridine, trichloroacetic acid, sodium chloride, potato starch, potassium and sodium tartrate tetrahydrate, sodium hydroxide and tris base were purchased from Fisher. Tetracosane, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu reagent, α-glucosidase from Saccharomyces cerevisiae, 4-nitrophenyl α-d -glucopyranoside (pNPG), lipase from porcine pancreas, colipase from porcine pancreas, 1,2-di-O-lauryl-rac-glycero-3-(glutaric acid 6-methylresorufin ester) (DGGR), angiotensin converting enzyme from rabbit lung (ACE) and N-[3-(2-furyl)acryloyl]-phe-gly-gly (FAPGG) were purchased from Sigma (St. Louis, MO, USA). N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), cholesterol, palmitic acid, 3,5-dinitrosalicylic acid, sodium deoxycholate, sodium taurodeoxycholate hydrate and captopril were purchased from Acros (Hampton, NH, USA). Pentadecanol and Orlistat were purchased from TCI (Tokyo, Japan). Sodium acetate and calcium chloride were purchased from ChemLab (Eernegem, Belgium). Acarbose was purchased from Fluka (Bucharest, Romania). All reagents were of analytical grade or of the highest available purity.
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