Dichloro dihydro fluorescein diacetate dcfh da

Acetonitrile, trifluoroacetic acid (TFA), ethanol, and methanol were obtained from Sigma-Aldrich (St. Louis, USA). HPLC-grade Acetonitrile and DMSO were obtained from Merck (Darmstadt, Germany). RPMI 1640, Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 (DMEM/F-12), Trypsin-EDTA, streptomycin, and penicillin were purchased from Caisson Lab (Utah, USA). Fetal bovine serum was supplied by Sigma-Aldrich (St. Louis, USA). Additionally, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), human insulin, epidermal growth factor, and hydrocortisone were purchased from Sigma (St. Louis, USA). Annexin-V-FITC Apoptosis Detection Kit and dichlorodihydrofluorescein diacetate (DCFH-DA) were also purchased from Sigma. JC-1 Mitochondrial Membrane Potential Assay Kit was procured from Calbiochem (California, USA). Additionally, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) was obtained from Thermo Fisher Scientific (MA, USA). All standard compounds including gallic acid (GA), ethyl gallate (EG), and penta-O-galloyl-β-D-glucose hydrate (PGG) were purchased from Sigma-Aldrich (St. Louis, USA).

Ethanol (99.80%), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Muller-Hinton agar (MHA), Muller-Hinton broth (MHB), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), and fetal bovine serum (FBS) were received from HiMedia, Mumbai, India. Calcium nitrate tetrahydrate, orthophosphoric acid, gallic acid, catechin, rutin, Dulbecco’s modified Eagle’s medium (DMEM), antibiotic and antimycotic solution, lipid peroxidation kit, Griess reagent, caspases kits (3/7, 8, and 9), antioxidant enzyme kits (SOD, CAT, and GSH), ELISA kits (TNF-α and IL-6), primer sequences (TNF-α, IL-6, iNOS, COX-2, and β-actin), dichloro-dihydro-fluorescein diacetate (DCFH-DA), TRI reagent, rhodamine 123, and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich, Bengaluru, India. The live/dead dual staining kit was purchased from Thermo Fisher Scientific and iScript One-Step RT-PCR kit with SYBR green was obtained from Bio-Rad, Bengaluru, India.

W 123 (10 µM), JTE 013 (1–10 µM), CAY 10444 (10 µM), specific inhibitors of S1PR1, S1PR2, S1PR3 respectively, S1P (0.1–10 µM), and the EGFR inhibitor tryphostin AG-1478 (0.3–3 µM) were all obtained from Cayman Chemical (Ann Arbor, MI, USA). Helenalin (1 µM), inhibitor of NF-κB, pEGFR antibody (p-Tyr-845 SC-23420-R) and total EGFR antibody (SC-03) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). SR 11302 (1 µM), inhibitor of activator protein-1 (AP-1) and SEW 2871 (10 µm), agonist of S1PR1 were obtained from Tocris Bioscience (Bristol, UK). Dichlorodihydrofluorescein diacetate (DCFH-DA) (10 µM) and N-acetyl cysteine (NAC) (1 mM) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Luciferase reporter lysis buffer was obtained from Promega (Madison, WI, USA). GM6001 (25 µM), the broad-spectrum hydroxamic acid inhibitor of matrix metalloproteinases (MMPs) and TAPI-1 (10 µM), inhibitor or MMPs and tumor necrosis factor-α converting enzyme (TACE) were obtained from Calbiochem (La Jolla, CA). Fura-2 AM (10 µM), and pluronic F127 (0.02%) were obtained from Life Technologies (Carlsbad, Ca).

Erythrocyte shape, eryptosis and intracellular reactive oxygen species (ROS) production in our different erythrocyte preparations (from human and zebrafish) were determined by flow cytometry using Beckman Coulter’s CytoFLEX and Cytexpert software (v2.1, Beckman Coulter, Brea, CA, United States). A specific erythrocyte cell population was selected by gating and could be characterized by its typical location in a forward scatter (FSC) versus a side scatter (SSC) parameter graph. For phosphatidylserine exposure determination, 100 µL of erythrocytes (1/50 dilution) were preliminary incubated with 2 μg/mL Annexin V-FITC in binding buffer (BioLegend, San Diego, CA, United States) for 30 min at RT before flow cytometry analysis. Annexin V protein exhibits a high affinity for phosphatidylserine (PS) and was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. For the evaluation of ROS production, 100 µL of erythrocytes (1/50 dilution) were incubated with 2 µM of fluorescent probes, dichlorodihydrofluoresceindiacetate (DCFH-DA; Sigma-Aldrich, Darmstadt, Germany) or dihydroethidium (DHE; Sigma-Aldrich, Darmstadt, Germany) for 30 min at RT.

Methanol, n-hexane, n-butanol, ethyl acetate, chloroform, Triton X-100 and Tween 20 were purchased from Fisher Scientific (Loughborough, Leicestershire, UK). Linoleic acid, gallic acid, quercetin, β-carotene (Type I synthetic, 95%), bovine serum albumin (BSA), ABTS, potassium persulfate, ferrous sulfate, hydrogen peroxide, AAPH, tetramethylchroman-2-carboxylic acid (trolox), ethylenediamine tetra acetic acid (EDTA), DPPH, dichloro-dihydro-fluorescein diacetate (DCFH-DA), Folin–Ciocalteu’s phenol reagent and ferrozine were supplied by Sigma-Aldrich Co. (St. Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), gentamicin and fetal bovine serum (FBS) were purchased from GIBCO (Grand Island, NY, USA). The 8-iso prostaglandin F2α ELISA kit was bought from Cusabio (Wuhan, China). MTT and neutral red were obtained from Nacalai Tesque (Kyoto, Japan). All of the other chemicals were purchased from Sigma-Aldrich Co.