N acetyl l cysteine

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N-acetyl-l-cysteine is a chemical compound that is commonly used in laboratory settings. It serves as a precursor to the antioxidant glutathione and has been studied for its potential therapeutic applications. The core function of N-acetyl-l-cysteine is to provide a source of the amino acid cysteine, which can be utilized in various biochemical processes.

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L-cysteine (59 citations) Cysteine (21 citations) N-acetyl-L-cysteine (NAC) (8 citations)

17 protocols using n acetyl l cysteine

Isolation and Culture of Retinal Ganglion Cells

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P3/P4 retinas were freshly dissected and RGCs were isolated and purified (>99%). For details see Steinmetz et al. (2006) (link); Claudepierre et al., 2008 (link)). Briefly, cells were cultured in Neurobasal medium (Gibco/Invitrogen) supplemented with (all from Sigma, except where indicated) pyruvate (1 mM), glutamine (2 mM; Gibco/Invitrogen), N-acetyl-l-cysteine (60 μg ml⁻¹), putrescine (16 μg ml⁻¹), selenite (40 ng ml⁻¹), bovine serum albumin (100 μg ml⁻¹; fraction V, crystalline grade), streptomycin (100 μg ml⁻¹), penicillin (100 U ml⁻¹), triiodothyronine (40 ng ml⁻¹), holotransferrin (100 μg ml⁻¹), insulin (5 μg ml⁻¹) and progesterone (62 ng ml⁻¹), B27 (1:50, Gibco/Invitrogen), brain-derived neurotrophic factor (BDNF; 25 ng ml⁻¹; PeproTech, London, UK), ciliary neurotrophic factor (CNTF; 10 ng ml⁻¹; PeproTech) and forskolin (10 μm; Sigma). After isolation, RGCs were either treated for RNA extraction or fixed with PFA4% 15' at RT and processed for immunohistochemistry. Stainings were performed and cells were visualized as described above.

Savier E., Eglen S.J., Bathélémy A., Perraut M., Pfrieger F.W., Lemke G, & Reber M. (2017). A molecular mechanism for the topographic alignment of convergent neural maps. eLife, 6, e20470.

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Apoptosis regulation by SphK1/2 inhibitors

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Chemical and reagents. Culture medium, N-Acetyl-L-cysteine (NAC) and ATP, fetal bovine serum (FBS) and antibiotics as well as the caspase-3 inhibitor z-DEVD-fmk and the pan caspase inhibitor z-VAD-fmk were from Invitrogen (Suzhou, China). Poly-d-Lysine was from BD Biosciences (Shanghai, China). Cell counting kit -8 (CCK-8) was provided by Dojindo Co. (Kumamoto, Japan). EdU (5-Ethynyl-2’-deoxyuridine) and TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) and JC-1 (5,5’,6,6’-Tetrachloro-1,1’,3,3’-tetraethyl-imidacarbocyanine),were from Thermo-Fisher Invitrogen (Shanghai, China). Antibodies for SphK1 (#12071), SphK2 (#32346), cleaved caspase antibody sampler kit (#9929) and β-Tubulin (#2146) were purchased from Cell Signaling Technologies (Beverly, MA). SP600125 and JNK inhibitor II (JNKi-II) were obtained from Selleck (Shanghai, China).

Jin L., Zhu J., Yao L., Shen G., Xue B.X, & Tao W. (2023). Targeting SphK1/2 by SKI-178 inhibits prostate cancer cell growth. Cell Death & Disease, 14(8), 537.

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Quantifying Intracellular ROS in Melanoma Cells

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2,7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA; Sigma, USA) was used to measure intracellular ROS production (17 (link)). A375 and C32 cells (1×10⁶ cells) were seeded onto a 12-well plate, and then treated with 5 mmol/L N-acetyl-L-cysteine (Invitrogen, USA) for 1 h. The cells with cultured with neferine (2.5, 5, or 10 µM); 6 h later, the medium was replaced with serum-free medium, and 10 µM DCFH-DA was added to the incubated cells for 20 min at 37 °C under dark conditions. Finally, the 2 cells were washed with phosphate-buffered saline (PBS), and dichlorofluorescein fluorescence was immediately assessed by a flow cytometer (Thermo Fisher, Waltham, MA, USA) and analyzed using FlowJo software (BD, Franklin Lakes, NJ, USA).

Xie J., Chen M.H., Ying C.P, & Chen M.Y. (2020). Neferine induces p38 MAPK/JNK1/2 activation to modulate melanoma proliferation, apoptosis, and oxidative stress. Annals of Translational Medicine, 8(24), 1643.

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Retinal Ganglion Cell Isolation and Purification

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P1/P2 retinas were freshly dissected and RGCs were isolated and purified (>99%). For details, see Steinmetz et al., 2006 (link) and Claudepierre et al., 2008 (link). Briefly, cells were harvested in neurobasal medium (Gibco/Invitrogen) supplemented with (all from Sigma, except where indicated) pyruvate (1 mM), glutamine (2 mM; Gibco/Invitrogen), N-acetyl-l-cysteine (60 mg/ml), putrescine (16 mg/ml), selenite (40 ng/ml), bovine serum albumin (100 mg/ml; fraction V, crystalline grade), streptomycin (100 mg/ml), penicillin (100 U/ml), triiodothyronine (40 ng/ml), holotransferrin (100 mg/ml), insulin (5 mg/ml) and progesterone (62 ng/ml), B27 (1:50, Gibco/Invitrogen), brain-derived neurotrophic factor (BDNF; 25 ng/ml; PeproTech, London, UK), ciliary neurotrophic factor (CNTF; 10 ng/ml; PeproTech) and forskolin (10 mM; Sigma). After isolation, RGCs were treated for RNA extraction.

Savier E.L., Dunbar J., Cheung K, & Reber M. (2020). New insights on the modeling of the molecular mechanisms underlying neural maps alignment in the midbrain. eLife, 9, e59754.

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Culturing Wnt3a/R-spondin/Noggin Organoids

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Mouse l-cells that expressed Wnt3a, R-spondin, and Noggin were commercially purchased (ATCC CRL-3276, Genova, Italy), and a conditioned medium (L-WRN) was prepared according to the instructions and protocol of the manufacturer. Culture medium with supplements (CM-S) was prepared using 50% conditioned L-WRN medium and 50% fresh primary culture media: Advanced DMEM/F-12 (cat.12634-010, Invitrogen, Milan, Italy) 1 mM N-Acetyl-l-cysteine (cat. A7250, Sigma, Milan, Italy), 1 × B-27^® supplements (cat.12587-010 Gibco, Milan, Italy), 50 ng/mL Epidermal Growth Factor (cat. PMG8041 Gibco Milan, Italy), 10 mM nicotinamide (cat. N0636, Sigma, Milan, Italy), 10 nM Leu15-gastrin I (cat. G9145 Sigma, Milan, Italy), 500 nM A8301 (inhibitor for ALK4/5/7, cat. 70024-90-7 Sigma, Milan, Italy), 10 µM SB202190 (p38 MAP kinase inhibitor, cat. S7076 Sigma, Milan, Italy), and 10 µM Y-27632 (p160 ROCK inhibitor; cat.1254 Tocris, Milan, Italy) in accordance with the protocols [45 (link),46 (link),47 (link)]. The organoids were cultured with a 300 µL culture medium, which was changed every second or third day.

Conte M., Nigro F., Porpora M., Bellomo C., Furone F., Budelli A.L., Nigro R., Barone M.V, & Nanayakkara M. (2022). Gliadin Peptide P31-43 Induces mTOR/NFkβ Activation and Reduces Autophagy: The Role of Lactobacillus paracasei CBA L74 Postbiotc. International Journal of Molecular Sciences, 23(7), 3655.

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Hepatoprotective Effects of DHI

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DHI was purchased from Shandong Buchang Pharmaceutical Co., Ltd (Jinan, China) (drug approval number: Z20026866). LPS from Escherichia coli 0111:B4 was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). D-galactosamine hydrochloride (GalN, 98%) and N-acetyl-L-cysteine (NAC, 98%) were purchased from Alfa AesarCo. (Tianjin, China). Detection kits for alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutathione S-transferase (GST), and total bilirubin (TBil) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The lipid peroxidation malondialdehyde (MDA) assay kit was purchased from Beyotime Institute of Biotechnology (China). Tumor necrosis factor (TNF)-α Mouse ELISA Kit was obtained from e-Bioscience Co. (USA). The DeadEnd Colorimetric TUNEL System and ImProm-II Reverse Transcription System were purchased from Promega Co. (USA). The Mammalian Cell Lysis Kit and UNIQ-10 column Trizol total RNA extraction kit were bought from Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). The FastStart Universal SYBR Green Master (ROX) kit was purchased from Roche (Mannheim, Germany).

Wang Y., Gao L.N., Cui Y.L, & Jiang H.L. (2014). Protective Effect of Danhong Injection on Acute Hepatic Failure Induced by Lipopolysaccharide and D-Galactosamine in Mice. Evidence-based Complementary and Alternative Medicine : eCAM, 2014, 153902.

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Multiparametric Flow Cytometry of Murine Immune Cells

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Single cell suspensions were stained for flow cytometry analysis using the following fluorochrome-conjugated antibodies specific to murine CD4 (GK1.5), CD8 (53–6.7), CD90.1 (OX-7), CD45R/ B220 (RA3-6B2), CD19 (6D5), CD262 (DR5) (MD5-1), CD253 (TRAIL) (N2B2), CD44 (IM7), CD62L (MEL-14), PD1 (RMP1-14), CD11b (M1/70), CD45.1 (A20), CD45.2 (104), anti–IL-2 (JES6-5H4), anti–TNF-α (MP6-XT22), anti–IFN-γ (XMG1.2), anti–GM-CSF (MP1-22E9), anti-GzmB (GB11), and antibodies specific to human CD4 (OKT4), CD8a (HIT8a) and CD262 (DR5) (DJR2-4). Antibodies were purchased from Biolegend unless otherwise noted. Anti-mouse FOXP3 (FJK-16s), and BCL-2 (Bcl2/100) were purchased from eBioscience. MCL-1 (B-6) (sc-74436) and Anti-FLIPS/L Antibody (G-11) (sc-5276) were purchased from Santa Cruz Biotechnology. Anti-COX2 (#10010096) antibody was purchased from Cayman Chemical. Polyclonal mouse anti-rat F(ab′)2 antibody and isotype control antibody were purchased from Jackson Immuno Research. Cyclophosphamide (CTX) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Indomethacin was purchased from Sigma-Aldrich. N-Acetyl-L-cysteine was purchased from Alfa Aesar. DR5 agonist antibody (MD5-1) was purchased from Bio X cell. Lipofectamine 2000 was purchased from ThermoFisher Scientific. Retronectin was purchased from Takara. D-Luciferin monosodium salt was purchased from Pierce.

Aboelella N.S., Brandle C., Okoko O., Gazi M.Y., Ding Z.C., Xu H., Gorman G., Bollag R., Davila M.L., Bryan L.J., Munn D.H., Piazza G.A, & Zhou G. (2022). Indomethacin-induced oxidative stress enhances death receptor 5 signaling and sensitizes tumor cells to adoptive T-cell therapy. Journal for Immunotherapy of Cancer, 10(7), e004938.

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Analytical Standards Chemical Analysis

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All the
reagents were of analytical grade
unless otherwise stated. Hydrogen tetrachloroaurate trihydrate (min.
99.9%), PESTANAL analytical standards of dithiocarbamate fungicides
(Thiram and Propineb) and organophosphate pesticides (Phorate and
Methamidophos), zofenopril calcium, arginine, asparagine, aspartic
acid, l-cysteine, cystine, dl-homocysteine, glutamine,
glutamic acid, ammonium chloride, lactic acid, magnesium sulfate,
magnesium chloride hexahydrate, sodium sulfide, sodium chloride, sodium
sulfate, sodium bicarbonate, dipotassium hydrogen phosphate, potassium
dihydrogen phosphate, citric acid, tri-sodium citrate, potassium chloride,
calcium chloride dehydrate, uric acid, d(+)-glucose, and
creatinine were obtained from Sigma-Aldrich (Steinheim, Germany). l-Glutathione (reduced), meso-2,3-dimercaptosuccinic acid, d-(−)-penicillamine, N-acetyl-l-cysteine, and captopril were obtained from Alfa Aesar (Karlsruhe,
Germany). Glycine, histidine, lysine, and valine were supplied by
Serva Electrophoresis GmbH (Heidelberg, Germany). Finally, urea (>99.5%)
was purchased from Pharmacia Biotech AB (Uppsala, Sweden) and HPLC-grade
solvent from Fischer Scientific (Loughborough, U.K.).

Kostara A., Tsogas G.Z., Vlessidis A.G, & Giokas D.L. (2018). Generic Assay of Sulfur-Containing Compounds Based on Kinetics Inhibition of Gold Nanoparticle Photochemical Growth. ACS Omega, 3(12), 16831-16838.

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Hepatocyte and Kupffer Cell Culture

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The hepatocyte culture medium was based on Williams' Medium E with GlutaMAX (Gibco, Paisley, UK), supplemented with 10% FCS (Gibco), 32 mU/mL Insulin (Sanofi Aventis, Frankfurt am Main, Germany), 15 mM HEPES, 0.1 mM MEM NEAA (100×), 1 mM pyruvate (all by Gibco), and 1 mg/L dexamethasone (Fortecortin, Merck, Darmstadt, Germany).
KC culture medium was based on RPMI low glucose (GE Healthcare, Pasching, Austria) supplemented with 10% FCS, 1% L-glutamine, and 6.3 mM N-acetyl-L-cysteine (all by Gibco). KC starvation medium was based on RPMI low glucose supplemented with 1% L-glutamine. All media were supplemented with 100 U/100 μM penicillin/streptomycin (Gibco) prior to use.
PBS was purchased from Gibco. Percoll, Trypan Blue, and Hanks Balanced Salt Solution (HBSS) were provided by Biochrom (Berlin, Germany). All other chemicals were purchased from Sigma (Munich, Germany), if not stated differently.

Kegel V., Pfeiffer E., Burkhardt B., Liu J.L., Zeilinger K., Nüssler A.K., Seehofer D, & Damm G. (2015). Subtoxic Concentrations of Hepatotoxic Drugs Lead to Kupffer Cell Activation in a Human In Vitro Liver Model: An Approach to Study DILI. Mediators of Inflammation, 2015, 640631.

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Doxorubicin Nanoparticle Cytotoxicity Assay

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Doxorubicin hydrochloride (DOX) was purchased from Sangon (Shanghai, China). Calcium chloride (CaCl 2 , 96%), disodium hydrogen phosphate (Na 2 HPO 4 , 99%) were obtained from Titan (Shanghai, China). Dopamine hydrochloride and 2-Amino-2-(hydroxymethyl)-1,3-propanediol (Tris) were obtained from Biofroxx (Guangzhou, China). FITC was purchased from Yeasen (Shanghai, China). Bovine serum albumin (BSA), 4′,6-diamidino-2-phenylindole (DAPI), 70 µm cell screen mesh, Live/Dead Viability/Cytotoxicity Kit (Promega), Advanced DMEM/F12 (Gibco), B-27 Supplement(50X), N2-supplement (100X), N-acetyl-L-cysteine, Recombinant Mouse EGF were purchased from Gibco (USA). Puromycin, Zeocin, Plasmocin, trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA) were gained from Invitrogen (USA). Matrigel Matrix was obtained from Corning (USA). CS10 was obtained from Stem Cell (Canada). CellTiter-Glo 3D Cell Viability Assay was purchased from Promega (USA). Rhodamine-phalloidin was purchased from Thermo Fisher (USA).

Deng T., Luo D., Zhang R., Zhao R., Hu Y., Zhao Q., Wang S., Iqbal M.Z, & Kong X. (2023). DOX-loaded hydroxyapatite nanoclusters for colorectal cancer (CRC) chemotherapy: Evaluation based on the cancer cells and organoids. SLAS technology, 28(1).

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