The following substances were used: 2,2-diphenyl-1-picrylhydrazyl, 5,5′-dithiobis (2-nitrobenzoic acid), bovine serum albumin, glutathione, butylated hydroxytoluene, Griess reagent, MTT, pyrogallol, xylenol orange (all from Sigma, St. Louis, USA), absolute ethanol, acetic acid, ascorbic acid, ferrous ammonium sulfate, hydrochloric acid, formaldehyde, hydrogen peroxide, methanol, sodium acetate, trichloroacetic acid (Vetec, Rio de Janeiro, RJ, Brazil), sulfuric acid, dimethyl sulfoxide and N,N-dimethylformamide (DMSO, Synth, Diadema, SP, Brazil), Dulbecco's Modified Eagle Medium (DMEM, Vitrocell, Campinas, SP, Brazil), fetal bovine serum (FBS, Gibco), and dextran sulfate sodium (Alfa Aesar, Heysham, Lancashire, UK).
Trichloroacetic acid
Manufactured by Vetec
Sourced in Brazil
Trichloroacetic acid is a colorless, crystalline compound with the chemical formula CCl3COOH. It is a strong organic acid commonly used in analytical chemistry and various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Xylenol orange, by Merck Group (3 mentions)
Absolute ethanol, by Vetec (3 mentions)
Methanol, by Vetec (2 mentions)
Pyrogallol, by Vetec (2 mentions)
Ferrous ammonium sulfate, by Vetec (2 mentions)
Tris hcl, by Merck Group (2 mentions)
K2hpo4, by Merck Group (2 mentions)
Kh2po4, by Merck Group (2 mentions)
Ethylenediaminetetraacetic acid, by Merck Group (2 mentions)
Formaldehyde, by Vetec (2 mentions)
Absolute methanol, by Vetec (2 mentions)
Dextran sulfate sodium (dss), by Thermo Fisher Scientific (1 mentions)
Sodium acetate, by Vetec (1 mentions)
Ascorbic acid, by Vetec (1 mentions)
Butylated hydroxytoluene, by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Glutathione (gsh), by Merck Group (1 mentions)
Griess reagent, by Merck Group (1 mentions)
Pyrogallol, by Merck Group (1 mentions)
7 protocols using trichloroacetic acid
1
Antioxidant and Cytotoxicity Assessment
2
Quantifying Total Cellular Glutathione
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To assess the total level of glutathione, the 3T3 cells (5 × 106 in six-well plate), obtained 24 h after oxidative stress induction, were washed with ice-cold 1× PBS, removed, resuspended in PBS and centrifuged (3000× g at 4 °C) twice for 5 min. After this process, the suspension obtained was then diluted in 50% trichloroacetic acid (Vetec, São Paulo, SP, Brazil) and centrifuged during 15 min (3000× g at 4 °C). After, the cell supernatant was diluted with the same volume 0.4 M Tris buffer contained 0.01 M dithiobisnitrobenzoic (Sigma-Aldrich, São Paulo, SP, Brazil). The material was read at 412 nm. The results were expressed as nmol/106 cells.
3
Antioxidant Evaluation of Phytochemicals
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Ouabain octahydrate, adenosine triphosphate (ATP), acetylthiocholine iodide, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dichlorofluorescein diacetate (DCFH-DA), 5,51-dithiobis-(2-nitrobenzoic acid)(DTNB), catechin, (−) epigallocatechin, (−) epigallocatechin gallate, quercetin, rutin, and kaempferol were acquired from Sigma Chemical Co. (St. Louis, MO, USA). Dibasic phosphate potassium (K2HPO4), monobasic phosphate potassium (KH2PO4), and trichloroacetic acid (TCA) were supplied by Vetec (Rio de Janeiro, RJ, Brazil). All chemicals and solvents were of analytical grade and the water used was glass distilled.
4
Glutathione Levels in 3T3 Cells
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To assess the total glutathione level, we cultured 3T3 cells (5 × 106 in a 6-well plate) for 24 h after inducing oxidative stress with H2O2, and L. ferrea EHMV was added at a concentration of 0.2 or 1.0 mg/mL. Subsequently, we washed and resuspended the cells in PBS and centrifuged them (3,000 × g at 4°C) twice for 5 min each. Next, we diluted the cell suspension in 50% trichloroacetic acid (Vetec, São Paulo, SP, Brazil) and centrifuged it for 15 min (3,000 g at 4°C). Next, we diluted the cell supernatant with an equal volume of 0.4 M Tris containing 0.01 M dithiobisnitrobenzoic acid (Sigma‒Aldrich, São Paulo, SP, Brazil). Subsequently, we measured the absorbance at 412 nm using a microplate reader (Epoch BioTek Instruments Inc., VT). The results are expressed in nmol/106 cells.
5
Ecobidens Glycolic Extract Anticancer Evaluation
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Ecobidens® (B. pilosa glycolic extract, BPE) and butylated hydroxytoluene were obtained from Chemyunion (Sorocaba, SP, Brazil) and Mapric (São Paulo, SP, Brazil), respectively. Polyethylene glycol l400, propylene glycol and sodium azide were acquired from Labsynth (Diadema, SP, Brazil). 5-Fluorouracil, hexadecyltrimethylammonium bromide, poloxamer 407, bovine serum albumin (BSA), n-butanol and ortho-dianisidine were purchased from Sigma–Aldrich (St. Louis, MO, USA). ImmunoCruz™ mouse ABC staining systems (sc-2017 and 2018), monoclonal mouse anti-mouse p53 (clone 3H2820) and polyclonal rabbit anti-mouse Bax (clone P-19) antibodies were acquired from Santa Cruz Biotechnology (CA, USA), whereas 3,3 diaminobenzidine (DAB), Duet strept ABC complex/HRP kit 0492 and anti-human Bcl-2 (clone 124) antibodies were obtained from Dako (Carpinteria, CA, USA). Anti-human ki-67 (clone MM1) antibodies were acquired from Novocastra (Newcastle, UK). Trichloroacetic acid, hydrogen peroxide (H2O2), tris, hydrochloricacid (HCl), methanol and xylene were obtained from Vetec (Rio de Janeiro, RJ, Brazil). Thiobarbituric acid, hematoxylin and eosin staining were acquired from Merck (Darmstadt, HE, Germany). Xylazine and ketamine were purchased from Syntec (Cotia, SP, Brazil) and König (Santana de Parnaíba, SP, Brazil), respectively.
6
Oxidative Stress Quantification Protocol
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Bovine serum albumin, 5,5′-dithiobis(2-nitrobenzoic acid), reduced glutathione (GSH), xylenol orange, K2HPO4, KH2PO4, 1 M Tris, 5 mM ethylenediaminetetraacetic acid, Tris HCl (all from Sigma, St. Louis, MO, USA), pyrogallol, absolute ethanol, absolute methanol, ferrous ammonium sulfate, trichloroacetic acid, formaldehyde (all from Vetec, Rio de Janeiro, Brazil), and ultra-pure water from a Milli-Q system were used for eluent preparation.
7
Antioxidant Capacity Quantification
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Bovine serum albumin, 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), reduced glutathione (GSH), xylenol orange, K2HPO4, KH2PO4, 1 M Tris, ethylenediaminetetraacetic acid, Tris HCl (all from Sigma, St. Louis, MO, United States), pyrogallol, absolute ethanol, absolute methanol, ferrous ammonium sulfate, trichloroacetic acid, formaldehyde (all from Vetec, Rio de Janeiro, Brazil), and ultra-pure water from a Milli-Q system were used for eluent preparation.
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