Polyethylene glycol

The following reagents were manufactured by Sigma Aldrich, USA: Iron(III) acetylacetonate (≥97.0%), Triethylene glycol (TEG) (99%), Sodium nitrite (NaNO₂) (≥97.0%), Sodium hydrocarbonate (NaHCO₃) (≥99.7%), Sulfuric acid (H₂SO₄) (95–98%), Phosphate-buffered saline (PBS), Dopamine hydrochloride (MQ 200), 3,3′-dithiodipropionic acid NHS ester (NHS-DTDP), N-Hydroxysuccinimide (NHS) (≥ 97.0%), Polyethylene glycol-acetic acid 2-aminoethyl ester, M = 3500 g/mol (NH₂-PEG-COOH), α,ω -bis{2-[(3-carboxy-1-hydroxypropyl)amino]ethyl}Polyethylene glycol, M = 3000 g/mol (PEG-COOH), N,N-Dimethylformamide (DMF) (≥99.8%), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (≥97.0%), N,N-Diisopropylethylamine (DIPEA) (≥99%), Dithiothreitol (DTT) (≥95%), 3,3′-dithiodipropionic acid (DTDP) (≥99%), Dimethyl sulfoxide (DMSO) (≥99%), and Epon resin. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTS) was manufactured by Promega, USA. Paraformaldehyde OH(CH₂O)nH (n = 8–100) was manufactured by Biovitrum, Russia. Cy5 NHS-ether and Cy3 amine dyes were manufactured by Lumiprobe, Russia. Ethanol (95%) was manufactured by Komponent-reactive, Russia. Deionized water was obtained in the Laboratory of Biomedical nanomaterials, NUST MISIS, Russia.

Walking-like activity was induced by continuous perfusion (20 ml/min) of D-glutamic acid (0.5 mM, Sigma, St. Louis, MO) or by NMDA (50 μM, Sigma, St. Louis, MO) with 10 μM D-serine (Sigma, St. Louis, MO) as described previously [14 (link)–15 (link), 17 (link)–19 (link)]. Individual gap junction uncoupling agents were added to the D-glutamic acid or NMDA solutions after the walking-like activity stabilized. Dose response curves were generated for four different uncoupling agents, carbenoxolone (CBX, 0.01–2 mM), flufenamic acid (FFA, 0.01–2 mM), 1-Heptanol and 1-Octanol (0.01–3 mM) (Sigma, St. Louis, MO). Stock solutions of CBX, 1-Heptanol, and 1-Octanol were prepared in Ringer’s solution. These blockers are less toxic than other compounds and they are water soluble or form emulsions in 25% ethylene glycol reducing the need for solvents such as DMSO and ethanol that affect the excitability of spinal neurons [19 (link), 31 (link), 32 (link)]. FFA was dissolved in 25% polyethylene glycol (Sigma, St. Louis, MO), as it has been shown that polyethylene glycol is not neurotoxic but neuroprotective [33 (link)]. All drugs were diluted in 100–200 ml of Ringer’s solution immediately before use. Drugs were washed out after each application with Ringer’s solution for at least 30 min before the next drug application.

2,2,2-Trifluoroethyl methacrylate (TFEMA, 99%), poly(ethylene glycol) behenyl ether methacrylate solution (PEGBEM, M_n ≈ 1500 g mol⁻¹, 50 wt.% in methacrylic acid/water), poly(vinylidene fluoride) (PVDF, M_w ≈ 534,000 g mol⁻¹), poly(ethylene glycol) (PEG, M_n = 400 g mol⁻¹), and bovine serum albumin (BSA, pH 7, ≥98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2,2′-Azobis(2-methylpropionitrile) (AIBN, 98%) was purchased from Acros Organics (Geel, Belgium) and used as the initiator for free-radical polymerization. All chemicals were used as received without further purification.

Bovine albumin, dextran, polyvinylpyrrolidone, polyethylene glycol, Triton-X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether), mannit, NaCl, KCl, MgCl₂, and phosphate buffer were purchased from Sigma Chemicals Co., St. Louis, MO, USA. The citrate–phosphate (McIlvaine) buffer (0.15 M, pH 5.0) was prepared by dissolving 1.815 g of Na₂HPO₄·2H₂O and 0.9605 g of citric acid to 100 mL dH₂O and adjusting the pH of the obtained solution to pH 5.0 by addition of HCl or NaOH.

Ixazomib was received as a gift from Takeda Pharmaceuticals U.S.A., Inc. MWCNTs with  > 98% carbon basis, O.D. × L 6–13 nm × 2.5–20 μm, size 10 μm length, and 12 nm outer diameter, (CAS no: 308068-56-6) were purchased from Sigma Aldrich and Graphite powder (< 20 μm, 99.995 + % purity, 45Im, B. No:282863 Aldrich). Polyethylene glycol (Mw = 6000 g mol⁻¹) Acid nitric (HNO₃), tetrahydrofuran (THF), dimethylformamide (DMF), triethylamine (TEA), sodium hydroxide (NaOH), hydrochloric acid (HCl), sulfuric acid (H₂SO₄), oxalyl chloride (COCl₂) B.NO: 08801 was purchased from Sigma-Aldrich and received from Acros Organics Co., Ltd. Methanol, acetonitrile, potassium chlorate, and all other chemicals and reagents were also purchased from Sigma-Aldrich (St. Louis, MO, USA).

C3H/HeJJmsSlc-lpr/lpr mice (Japan SLC, Shizuoka, Japan) were injected intraperitoneally with an emulsion containing 0.2 mg of oxidized-form HD5 and Freund’s complete adjuvant. After 2 weeks, an emulsion containing 0.2 mg of oxidized-form HD5 and Freund’s incomplete adjuvant. Two weeks later, mice were injected intraperitoneally with 0.2 mg of oxidized-form HD5 dissolved in phosphate-buffered saline (PBS) as the last booster injection. After the final injection, mice were euthanized and splenocytes were obtained. Hybridomas were produced by fusing the splenocytes with mouse myeloma P3U1 cells in the presence of 50% polyethylene glycol (Sigma-Aldrich, St. Louis, MO). Hybridoma clones producing oxidized-form HD5-specific antibody were screened by indirect enzyme-linked immunosorbent assay (ELISA) as described previously [47 (link)] and subcloned four times by the limiting dilution method. Reactivities of each clone pair against oxidized-form HD5 were evaluated by comparing the sensitivities of sandwich ELISA, and the pair of clones 12-1 and 39E-7 showing the highest sensitivity against oxidized-form HD5 was finally selected for the establishment of sandwich ELISA.