The following standards were acquired from Sigma-Aldrich (Steinheim, Germany): glucose, sucrose, fructose, inositol, spermidine, spermine, cadaverine, putrescine, 1,6-hexaendiamine gallic acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS•+), and benzoyl chloride. Sodium hydroxide was purchased from Fluka (Buchs, Switzerland), and LC-MS grade organic solvents (Methanol, acetonitrile), together with sodium carbonate, Folin-Ciocalteu reagent, and ethyl ether, were purchased from Panreac Química (Barcelona, Spain). SPE cartridges (C18 Sep-Pak cartridges) were obtained from Waters Associates (Milford, MA, USA), and, lastly, ultrapure water was produced by a Millipore water purification system.
Inositol
Manufactured by Merck Group
Sourced in United States, Germany, China, France
Inositol is a naturally occurring sugar alcohol found in various plant and animal tissues. It plays a crucial role in cellular function and signaling processes. Inositol is commonly used as a laboratory reagent and research tool to study these cellular mechanisms.
Automatically generated - may contain errors
Lab products found in correlation
Folic acid, by Merck Group (28 mentions)
Horse serum, by Thermo Fisher Scientific (14 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (13 mentions)
2 mercaptoethanol, by Merck Group (12 mentions)
L glutamine, by Thermo Fisher Scientific (12 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (8 mentions)
Alpha minimum essential medium, by Thermo Fisher Scientific (8 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (7 mentions)
Sucrose, by Merck Group (5 mentions)
Sodium bicarbonate, by Merck Group (5 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (5 mentions)
α mem, by Thermo Fisher Scientific (5 mentions)
Nk 92mi, by Thermo Fisher Scientific (5 mentions)
Fructose, by Merck Group (4 mentions)
Arabinose, by Merck Group (4 mentions)
Galactose, by Merck Group (4 mentions)
Folic acid, by Thermo Fisher Scientific (4 mentions)
Horse serum, by Merck Group (4 mentions)
Nk 92mi, by American Type Culture Collection (4 mentions)
Recombinant il 2, by Thermo Fisher Scientific (4 mentions)
60 protocols using inositol
1
Analytical Standards for Metabolite Quantification
2
Analytical Reference Compounds for Chromatography
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Analytical and chromatographic grade reference compounds were used for this study: Xylose, arabinose, glucose, galactose, xylitol, mannitol, sorbitol, inositol, ribose, fructose, mannose, adonitol, sucrose, maltose, lactose, and maltitol were purchased from Sigma-Aldrich GmbH (Steinheim, Germany). HPLC grade acetonitrile was obtained from Sigma-Aldrich GmbH (Steinheim, Germany), and purified deionized water (18.2 mW/cm) was produced using the Millipore (Millipore, Bedford, MA, USA) water purification system.
3
Culturing Diverse Hematological Cell Lines
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DC4 + human primary AML samples and normal peripheral blood mononuclear cells (PBMCs) were obtained from residual samples using a protocol approved by the Institutional Review Board of Stony Brook University. THP-1, U937, TALL104, and NK-92 cell lines were obtained from ATCC (Manassas, VA, USA). MOLM-13 was obtained from AddexBio (San Diego, CA, USA) T cells were cultured in filtered T cell media, defined as 50% AIM V, 40% RPMI 1640 and 10%FBS, with 1% Pen/Strep (all Gibco, Waltham, MA, USA) and supplemented with IL-2 (300 IU/mL; Peprotech, Rocky Hill, NJ, USA), unless otherwise specified. NK-92 cells were cultured in filtered NK cell media, defined as alpha-MEM without ribonucleosides and deoxyribonucleosides with 2mM L-glutamine, 1.5 g/L sodium bicarbonate (Gibco), 12.5% heat-inactivated horse serum (Gibco), 12.5% heat-inactivated FBS (Atlanta Biologicals, Atlanta GA, USA), 1% Pen/Strep (Gibco), 0.2% inositol (Sigma), 0.02% folic acid (Fisher), and 50 uM beta-mercaptoethanol (Fisher), supplemented with IL-2 (300 IU/mL), unless otherwise specified. THP-1, U937, and MOLM-13 cell lines were cultured in RPMI, 10% FBS, 1% Pen/Strep (Gibco). TALL104 cells were cultured in IMDM adding 300 IU/ml recombinant human IL-2, 2.5 mg/ml human albumin, 0.5 mg/ml D-mannitol, and 20% FBS.
4
Culturing and Treating NK-92MI Cells
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NK-92MI cells were cultured at 37 °C with 5% CO2 in alpha minimum essential medium (αMEM, Gibco, Waltham, MA, USA) supplemented with 12.5% horse serum and 12.5% fetal calf serum (FCS) (Gibco), containing 0.2 mM inositol (Sigma, Le Chesne, France), 0.1 mM 2-β mercaptoethanol (Gibco), 0.02 mM folic acid (Sigma), 100 U/mL penicillin, and 100 µg/mL streptomycin. To evaluate the effect of different serum additives, we added 10% FCS or 10% human AB serum (ABS) (Gibco) to the serum-free culturing medium and incubated the cell for 24 h at 37 °C. Treatment with human E. coli-derived recombinant Galectin-9 (Gal-9, Cat No:20-45GA, R&D Systems, Minneapolis, MN, USA) was performed in 30 and 100 nM concentrations for 24 h at 37 °C. K562 cells were maintained in Roswell Park Memorial Institute 1640 medium (RPMI, Lonza, Verviers, Belgium) supplemented with 10% FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin (Lonza).
5
Culturing Human Cancer and NK Cell Lines
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The human tumor cell line K562 (human chronic myelogenous leukemia, CML) and A2780 (human ovarian cancer cell line) were obtained from the ATCC. Cells were cultured in RPMI 1640 medium (Gibco/Life Technologies, Carlsbad, CA) supplemented with 10% heat-inactivated FBS, 200 mM L-glutamine and 10,000 U/mL Pen/Strep (Gibco/Life Technologies, Carlsbad, CA) at 37 °C in a 5% CO2 incubator. NK-92, the human NK cell line, was purchased from the ATCC and cultured in Alpha MEM devoid of ribonucleosides and or deoxyribonucleosides, 12.5% FBS, 10,000 U/mL Pen/Strep (Gibco/Life Technologies, Carlsbad, CA), 2 mM L-glutamine (Gibco/Life Technologies, Carlsbad, CA), 0.2 mM inositol (Sigma-Aldrich, St. Louis, MO), 0.02 mM folic acid (Sigma-Aldrich, St. Louis, MO), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO), and 100–200 U/ml recombinant IL-2 (Peperotech, Inc., NJ).
6
Sugars Extraction and Composition Analysis
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The sugars were obtained by repeated four-time extraction with boiling water for 20 min. Proteins were removed from the resulting extract [70 ]. The extract was further purified of charged compounds using a combined column with the Dowex-1 (acetate form) and Dowex 50 W (H+) ion exchange resins. The sugar composition was determined by gas-liquid chromatography using trimethylsilyl sugar derivatives obtained from the lyophilized extract [71 ]. α-Methyl-D-mannoside (“Merck”, Darmstadt, Germany) served as the internal standard. Chromatography was performed with a Kristall 5000.1 gas chromatograph (“Chromatek”, Yoshkar-Ola, Russia) equipped with a ZB-5 30 × 0.32 mm capillary column (“Phenomenex”, Torrance, CA, USA) temperature program was set at +130, 5–6 °C/min gradient to +270 °C. Glucose, mannitol, arabitol, inositol, and trehalose (“Sigma”, Sant Louis, MO, USA) served as the standards.
7
Cell Culture Protocols for Cancer and Immune Cell Lines
8
NK Cell Cultivation and Tumor Cell Lines
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NK-92 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, United States). NK-92 cells were maintained in α-Minimum Essential Medium (MEM) containing 12.5% fetal bovine serum (FBS; Welgene, Gyeonsan, Korea), 12.5% horse serum (Gibco, Waltham, MA, United States), 0.2 mM inositol (Sigma-Aldrich, St. Louis, United States), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich), 0.02 mM folic acid (Sigma-Aldrich) and 20 ng/μl IL-21 (PeproTech, Rocky Hill, United States).
K562 and YAC-1 cells, the human and mouse T cell lymphoma cells sensitive to NK cell-mediated lysis, were purchased from ATCC and cultured in RPMI containing 10% heat-inactivated FBS. The B16F10 murine melanoma cell line, which shows metastatic activity in the lungs of syngeneic C57BL/6 mice, was purchased from ATCC and cultured in DMEM containing 10% heat inactivated FBS. All tumor cell lines were cultured and maintained in a CO2 incubator at 37°C.
K562 and YAC-1 cells, the human and mouse T cell lymphoma cells sensitive to NK cell-mediated lysis, were purchased from ATCC and cultured in RPMI containing 10% heat-inactivated FBS. The B16F10 murine melanoma cell line, which shows metastatic activity in the lungs of syngeneic C57BL/6 mice, was purchased from ATCC and cultured in DMEM containing 10% heat inactivated FBS. All tumor cell lines were cultured and maintained in a CO2 incubator at 37°C.
9
Cell Culture and Maintenance Protocols
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NK cell line NKG was established and maintained in our labratory as previously described [29 ] and cultured in α‐MEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 15% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA), 15% horse serum (Thermo Fisher Scientific, Waltham, MA, USA) and 100 U/mL rhIL‐2 (Kingsley, Yixing, Jiangsu, China). NK cell line NK92 was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in α‐MEM supplemented with 12.5% FBS, 12.5% horse serum, 0.2 mmol/L inositol (Sigma Aldrich, St. Louis, MO, USA), 0.1 mmol/L 2‐mercaptoethanol (Sigma Aldrich, St. Louis, MO, USA), 0.02 mmol/L folic acid (Sigma Aldrich, St. Louis, MO, USA) and 100 U/mL rhIL‐2. The leukemia cell lines U‐937 and THP‐1 were obtained from ATCC and cultured in RPMI‐1640 medium (Hyclone, South Logan, UT, USA) containing 10% FBS. The erythroleukemia cell line K562 were obtained from National Collection of Authenticated Cell Cultures (Shanghai, China) and cultured in IMDM medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% FBS. The human embryonic kidney cell line HEK293T were obtained from National Collection of Authenticated Cell Cultures (Shanghai, China) and cultured in DMEM medium (Hyclone, South Logan, UT, USA) containing 10% FBS. All cell lines used in this study were maintained at 37°C in 5% CO2.
10
Comprehensive LC-MS Analytical Methodology
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LC-MS-grade acetonitrile and ammonium acetate, as well as NH3, were obtained from VWR (Vienna, Austria). Analytical-grade acarbose, arabinose, erythritol, fructose, galactose, glucose, inositol, lactitol, lactose, maltitol, raffinose, rhamnose, ribose, sucrose, xylitol, potassium chloride, ammonium iodide and potassium bromide were purchased from Sigma Aldrich (Schnelldorf, Germany). Erythrose, isomaltulose, lyxose, maltose, maltotriose, mannitol, mannose, sorbitol, sorbose, xylose, sodium nitrate and sodium sulfate were purchased from VWR (Vienna, Austria). LC-MS-grade water (< 0.055 µS cm−1) from an ultrapure water purification system (Sartorius, Göttingen, Germany) was used for both elution and sample preparation. Food samples produced by various companies were obtained from local supermarkets. Sample matrices have been selected over a wide range of beverages and food to investigate the impact on sample preparation and robustness of the analytical method. Food and beverages were stored at the recommended temperature until analysis.
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