Metaphosphoric acid

RNeasy kit, reverse transcription kit, and QuantiTect SYBR Green PCR kit were purchased from Qiagen Inc., Valencia, CA. qPCR primers were designed using Primer Bank (https://pga.mgh.harvard.edu/primerbank/) or Primer BLAST website and purchased from Integrated DNA Technologies, Coralville, IA. Trans-AM Nrf2 kit (50296) for determination of Nrf2-ARE binding activity was obtained from Active Motif, Carlsbad, CA. GCLC (ab41463), NQO1 (ab34173), GSR (ab16801), and Ubiquitin (ab7780) antibodies were procured from Abcam, Cambridge, MA; rabbit anti-GAPDH (D16H11) from Cell Signaling, USA. Anti-rabbit or mouse secondary antibodies for immunoblots (horse radish peroxidase conjugated with IgG) were purchased from Vector Laboratories, Burlingame, CA. Protein Assay reagent (#500–0006) was procured from Bio-Rad, Hercules, CA. All other chemicals including oxidized glutathione, RNAlater, meta-phosphoric acid, and isoproterenol, were purchased from Sigma-Aldrich unless otherwise stated.

As previously described, PBS perfused brain regions were frozen in liquid nitrogen then freeze fractured (Kaczmarczyk et al., 2013 (link)) in reaction buffer containing 50 mM NaCl (Fisher Scientific, Fair Lawn, NJ, USA), 1 mM EDTA, 50 mM HEPES, pH 7.0 (USB Corporation, Cleveland, OH, USA) using the TissueLyser II (Qiagen, Valencia, CA, USA) at a rotational frequency of 30 s⁻¹ for 2 min. Lysates were centrifuged at 10,000× g for 15 min at 4°C and the supernatant recovered. The supernatant was deproteinated with an equal volume of metaphosphoric acid (Sigma-Aldrich, St. Louis, MO, USA) and vortexing. Samples were re-centrifuged at 8000× g for 5 min. Supernatant and pellets were saved. Glutathione, both reduced and oxidized, was determined using the Glutathione Assay Kit (Cayman Chemical, Ann Arbor, Michigan) following the manufacturer’s instructions. Glutathione (GSH) and glutathione disulfide (GSSG) were quantified using an ELx800 Absorbance Microplate Reader (BioTek Instrument, Winooski, VT, USA) at 405 nm in 5 min intervals for 30 min. Protein precipitates were eluted with reaction buffer and quantified using the DC Protein Assay (Bio-Rad, Hercules, CA, USA).

Metaphosphoric acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH),NaOH, nitroblue tetrazolium, dichlorophenol-indophenol (DCPID),2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) di-ammoniumsalt (ABTS•+), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxyl acid(Trolox), phenazine methosulphate, ethanol, gallic acid, ethylenediaminetetraaceticacid (EDTA), ferrozine, 2,4,6-tris(2-piridil)-s-triazina(TPTZ) and iron sulphate heptahydrate were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Acetonitrile and acetic acid were HPLC-grade and were purchased from Merck (Darmstadt, Germany). All the phenolic standards were obtained from Extra Syntheses (Genay, France). Solvents and reagents for carotenoid detection were purchasedfrom Panreac (Barcelona, Spain). Other chemicals were of analyticalgrade purchased from Carlo Erba Reagents s.r.l. (Cornaredo, MI, Italy).

L-Ascorbic acid and metaphosphoric acid (MPA) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA) and Kanto Chemicals Co. Inc. (Tokyo, Japan), respectively. HPLC-grade acetonitrile and water were purchased from Fisher Scientific (Pittsburgh, PA, USA). All other chemicals were of analytical grade.

Methylglyoxal (MG), propidium iodide, [3(4,5-dimethylthi-azol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT), neutral red, carbenoxolone disodium salt (CBX), aminoguanidine hemisulfate salt (AG), standard glutathione, o-phthaldialdehyde, metaphosphoric acid, L-glutamate, N-methyl-D-glucamine, 4-(2-hydroxyethyl) piperazine-L-ethanesulfonic acid (HEPES), and cell culture materials were purchased from Sigma (Saint Louis, MO, USA). Dulbecco's modified Eagle medium (DMEM) and Dulbecco's phosphate-buffered saline (DPBS) were purchased from Gibco BRL (Carlsbad, CA, USA). Fetal bovine serum was obtained from Cultilab (Campinas, SP, Brazil), and L-[2,3-³H] glutamate was purchased from Amersham International (United Kingdom). Polyclonal anti-EAAT1 (GLAST) and anti-EAAT2 (GLT-1) were purchased from Abcam (Cambridge, MA, USA), anti-EAAT3 was purchased from Novus Biologicals (Littleton, CO, USA), and polyclonal anti-glyoxalase 1 was purchased from Santa Cruz Biotechnology Inc. (Dallas, Texas, USA). All other chemicals were purchased from local commercial suppliers.

Phosphate-buffered saline (PBS), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and antibiotics (amphotericin B, penicillin, and streptomycin) were purchased from Invitrogen (Carlsbad, CA, USA). Folin reagent, (+)-catechin, sodium carbonate, gallic acid, metaphosphoric acid, and other chemicals were obtained from Sigma (St Louis, MO, USA). The pro-collagen type I (cat#. ab210966) and MMP-1 (cat#. ab100603) enzyme-linked immunosorbent assay (ELISA) kit was obtained from Abcam (Cambridge, UK). The primary and secondary antibodies used in Western blot analyses were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). All other chemicals were of analytical grade or complied with the standards required for cell culture experiments.

We used o-phthalaldehyde (OPT) (Sigma-Aldrich) as the fluorescent reagent to
quantify the level of reduced glutathione due its specificity for GSH. A
total of 100 μL of cells in PBS supplemented with protease inhibitors and
100 μL of meta-phosphoric acid (Sigma-Aldrich) precipitating reagent (1.67 g
meta-phosphoric acid, 0.2 g EDTA (AMRESCO, Solon, OH, USA) and 30 g NaCl in
100 mL of distilled water) were added to a 0.6 mL Eppendorf tube, vortexed
and centrifuged. The supernatant was decanted and frozen at -70 °C prior to
further quantification. A total of 50 μL of the supernatant was added to a
1.5 mL Eppendorf tube with 1 mL of GSH buffer (0.1 M
NaH₂PO₄ and 0.005 M EDTA, pH 8.0). Then, 50 μL of
OPT (1mg/mL in methanol) was added to obtain a GSH-fluorescent conjugate.
Next, 200 μL of the mixture from each Eppendorf tube was plated in an opaque
96-well plate and incubated for 15 min in the dark. The fluorescence was
read in a BioTek FLx800 Fluorescence Microplate Reader (Winooski, VT, USA)
with the emission set at 420 nm and the excitation set at 350 nm (Browne and Armstrong, 1998 (link)).