4.5 g l glucose

Manufactured by Thermo Fisher Scientific

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4.5 g/L glucose is a laboratory reagent used as a standard solution in various analytical procedures. It provides a consistent concentration of glucose for calibration, quality control, and other experimental purposes.

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L-glucose (7 citations) DMEM with 4.5 g/L glucose (7 citations) DMEM) containing 4.5 g/L glucose (6 citations) DMEM (4.5 g/L d-glucose; (3 citations) DMEM) Glutamax 4.5 g/l glucose (2 citations) Glutamine and 4.5 g/L glucose (2 citations) Dulbecco's modified Eagle medium (DMEM with glucose 4.5 g L−1), (2 citations) Dulbecco’s modified Eagle’s high glucose w/Glutamax (4.5 g/L) cell culture medium (2 citations) GlutaMAX and 4.5 g/L glucose (2 citations) DEMEM High Glucose (4.5 g/l) medium (1 citations)

19 protocols using 4.5 g l glucose

Skeletal Muscle Explant Culture Protocol

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Skeletal muscle samples from animals injected with experimental plasmids were harvested at day 2 after injection and used for generation skeletal muscle explant culture. Skeletal muscle explant culture experiments were performed in accordance with the protocol described by Jang and Kim [24 (link)]. Skeletal muscle samples were placed in Matrigel and then cultured in complete DMEM with 4.5 g/L glucose (Life Technologies, USA) and 2% FBS (Gibco, USA) under standard conditions. Culture medium samples were harvested on the 3rd and 6th days in culture. HGF and VEGF165 concentrations were evaluated by ELISA as described above.

Slobodkina E., Boldyreva M., Karagyaur M., Eremichev R., Alexandrushkina N., Balabanyan V., Akopyan Z., Parfyonova Y., Tkachuk V, & Makarevich P. (2020). Therapeutic Angiogenesis by a “Dynamic Duo”: Simultaneous Expression of HGF and VEGF165 by Novel Bicistronic Plasmid Restores Blood Flow in Ischemic Skeletal Muscle. Pharmaceutics, 12(12), 1231.

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Autophagy Signaling Pathway Assay

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Antibodies to Akt, phospho-Akt (Ser473), phospho-Akt (Thr308), beclin-1, phospho-Beclin-1 (Ser15), phospho-PI3Kinase p85(Tyr458)/p55 (Tyr199), phospho-4E-BP1 (Thr37/46), p70S6K, phospho-p70S6K(Thr389), LC3B, poly(ADP-ribose) polymerase (PARP), cleaved PARP (D214) and anti-rabbit IgG HRP-linked antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse monoclonal β-Actin antibody and crude fucoidan from F. vesiculosus were obtained from Sigma-Aldrich (St Louis, MO, USA). DMEM (Dulbecco’s Modification of Eagle’s Medium) with 4.5 g/L glucose, L-glutamine and sodium pyruvate, trypsin-EDTA, penicillin-streptomycin-amphotericin B solution (50X), fetal bovine serum (FBS), phosphate buffer solution (PBS), and PBS with Tween 20 (PBST) were purchased from Life Technologies (Waltham, MA, USA).

Zhang J., Riby J.E., Conde L., Grizzle W.E., Cui X, & Skibola C.F. (2016). A Fucus vesiculosus extract inhibits estrogen receptor activation and induces cell death in female cancer cell lines. BMC Complementary and Alternative Medicine, 16, 151.

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Evaluating Digestibility of Sodium Caseinate

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Commercial sodium caseinate (Casinella-QN 94.2% protein Molkerei Meggle Wasserburg GMBH & Co. KG) was kindly donated by Kelta Ltd. Israel and Molkerei Meggle Wasserburg GMBH & Co. KG, Germany. VD 3 in powder (USP grade), potassium phosphate dibasic (USP), potassium citrate tribasic monohydrate (FCC) and calcium chloride dihydrate (FCC), pepsin (Sigma P7000, ≥250 units/mg protein), Trypsin (Sigma T0303, 13,000-20,0000 BAEE units/mg protein), chymoTrypsin (Sigma C4129, ≥40 units/mg protein), pancreatic lipase (Sigma L3126, 100-500 units/mg protein), sodium glycodeoxycholate (Sigma G9910) and taurocholic acid salt hydrate (Sigma T4009) were purchased from Sigma-Aldrich (Rehovot, Israel). Sodium chloride and Sodium bicarbonate were purchased from Frutarom Industries Ltd. (Herzliya, Israel) and Hydrochloric acid was purchased from Gadot group (Netanya, Israel). Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/L glucose, penicillin/streptomycin, Trypsin-EDTA (500 mg/L and 200 mg/L, respectively) and PBS were purchased from Life Technologies (Saint Aubin, France). Fetal bovine serum was obtained from PAA (Vélizy Villacourblay, France).

Cohen Y., Levi M., Lesmes U., Margier M., Reboul E, & Livney Y.D. (2017). Re-assembled casein micelles improve in vitro bioavailability of vitamin D in a Caco-2 cell model. Food & function, 8(6).

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Fibroblast Culture of Healthy and PARK6 Patients

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Human fibroblasts of 7 healthy controls and 3 PARK6 patients [17 (link)] (passage 8–12) were cultured in Dulbecco’s Modified Eagle Medium 4.5 g/L glucose (Invitrogen) plus 15% bovine growth serum (BGS, Thermo Scientific), 1% glutamine, 1% penicillin/streptomycin (all Invitrogen) at 37 °C and 5% CO₂ in a humidified incubator.

Key J., Sen N.E., Arsović A., Krämer S., Hülse R., Khan N.N., Meierhofer D., Gispert S., Koepf G, & Auburger G. (2020). Systematic Surveys of Iron Homeostasis Mechanisms Reveal Ferritin Superfamily and Nucleotide Surveillance Regulation to be Modified by PINK1 Absence. Cells, 9(10), 2229.

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Osteosarcoma Cell Line Cultivation

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The murine OS cell line LM8 (RCB1450), and human OS cell lines 143B (RCB0701), HOS (RCB0992), and MG63 (RCB1890) were purchased from Riken Cell Bank (Tsukuba, Japan). The human osteoblast cell line hFOB 1.19 (ATCC CRL-11372) was purchased from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in growth media consisting of Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 4.5 g/L glucose (Invitrogen, Carlsbad, CA, USA), 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO₂. All cell cultures were tested for mycoplasma contamination using a MycoAlert Mycoplasma Detection Kit (Lonza, Ann Arbor, MI, USA). Gem and Rapa were purchased from Sigma-Aldrich (St. Louis, MO, USA). The pan-caspase inhibitor Z-VAD-FMK was purchased from Merck Millipore (Darmstadt, Germany).

Ando T., Ichikawa J., Fujimaki T., Taniguchi N., Takayama Y, & Haro H. (2020). Gemcitabine and Rapamycin Exhibit Additive Effect against Osteosarcoma by Targeting Autophagy and Apoptosis. Cancers, 12(11), 3097.

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Chondrogenic Differentiation of TC-Expanded Cells

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Aggregates were made from TC-expanded cells as described previously (6 (link)). Briefly, 250,000 cells were dispensed into several wells of a sterilized (autoclaved) polypropylene v-shaped bottom 96-well plate (Phenix, Hayward, CA), centrifuged at 590 RCF for 10 min, and placed in chondrogenic medium (serum-free DMEM with 4.5g/L glucose, containing 1% sodium pyruvate, and 1% penicillin/streptomycin [all from Invitrogen], 1% ITS-premix [BD Bioscience], 100 nM dexamethasone [Sigma-Aldrich], and 37.5 µg/mL L-ascorbate-2-phosphate [Wako chemicals]. The aggregates were cultured under the same oxygen condition as that used in expansion, and medium was changed every other day. At 21 days, the aggregates were collected and processed for combined GAG and DNA analysis (3 aggregates from each group), for combined collagen and collagen cross-links analysis (3 aggregates from each group), for lysyl oxidase (LOX) activity assay (3 aggregates from each group), for LOX gene expression analysis (3 aggregates from each group, stored in RNA Later® [Qiagen] at −80°C), and fixed in neutral buffered formalin for histology (1 aggregate from each group). Wet weights of all aggregates were taken after blotting excess media using filter paper.

Kean T.J., Mera H., Whitney G.A., MacKay D.L., Awadallah A., Fernandes R.J, & Dennis J.E. (2016). Disparate Response of Articular- and Auricular-derived Chondrocytes to Oxygen Tension. Connective tissue research, 57(4), 319-333.

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Cisplatin-Resistant Colorectal and Cervical Cancer Cell Lines

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This study employed the DLD1 human colorectal carcinoma cells, parental human cervical carcinoma cell line KB-3-1 (a sub-line of HeLa), and its cisplatin-resistant sub-line KB-CP.5. KB-CP.5 cells were originally selected in a single step in 0.5 μg cisplatin/mL (1.6 μM) in our laboratory, as described previously (20 (link), 21 (link)). KB lines were originally generated in the laboratory of MMG. DLD-1 cells were provided by the National Cancer Institute (part of the NCI-60 collection). All cell lines were thawed immediately prior to experimentation, and cell lines are characterized by NCI using short tandem repeat profiling. The cisplatin stock solution used for culturing CP.5 cells were prepared in PBS. The cisplatin-resistant cells were maintained in the presence of cisplatin, which was removed from growth medium three days prior to all experiments. All cell lines were grown as monolayer cultures at 37°C in 5% CO₂, using either Dulbecco's modified Eagle medium (DMEM, KB cells), or Roswell Park Memorial Institute medium (RPMI, DLD1 cells) with 4.5 g/L glucose (both from Invitrogen, Carlsbad, CA), supplemented with L-glutamine, penicillin, streptomycin and 10% fetal bovine serum (BioWhittaker, Walkersville, MD). Resistance of CP.5 cells to cisplatin was confirmed on a regular (at least monthly) basis, using cell viability assays as described herein.

Hall M.D., Telma K.A., Chang K.E., Lee T.D., Madigan J.P., Lloyd J.R., Goldlust I.S., Hoeschele J.D, & Gottesman M.M. (2014). Say No to DMSO: Dimethylsulfoxide Inactivates Cisplatin, Carboplatin and Other Platinum Complexes. Cancer research, 74(14), 3913-3922.

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Hypoxia-Reoxygenation Rat Kidney Cell Assay

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The rat kidney tubular epithelial cell line (NRK-52E) was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were maintained in DMEM containing 4.5 g/L glucose (Invitrogen) with 10% FBS at 37°C in a 5% CO 2 atmosphere and passaged twice a week. Cells were plated in 35-mm dishes (Corning Inc., Corning, NY, USA) at a density of 1 × 10 6 cells/dish, reached ~90% confluence by the next day, and were used for experiments. For hypoxia experiments, cells were treated in a hypoxia C-chamber (BioSpherix, Lacona, NY, USA) inside a standard CO 2 incubator (Revco Ultima II; Thermo Fisher Scientific) with a compact gas oxygen controller (ProOx P110; BioSpherix) to maintain oxygen concentration at 1% by introducing a gas mixture of 95% N 2 and 5% CO 2 for 24 h. After hypoxic treatment, the cells were washed with PBS (Gibco) and cultured with NM or iPSC-CM in 95% O 2 /5% CO 2 condition for 6 h (reoxygenation). Control cells were incubated in a regular cell culture incubator with 21% O 2 . At the end of the study, cells were monitored morphologically or harvested with indicated buffers to collect cell lysates for biochemical analyses.

Tarng D.C., Tseng W.C., Lee P.Y., Chiou S.H, & Hsieh S.L. (2016). Induced Pluripotent Stem Cell-Derived Conditioned Medium Attenuates Acute Kidney Injury by Downregulating the Oxidative Stress-Related Pathway in Ischemia-Reperfusion Rats. Cell transplantation, 25(3).

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Cisplatin Treatment and Apoptosis Inhibition

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The lung adenocarcinoma cell line A549 (ATCC, CCL-185), lung large cell carcinoma U1810 (CVCL D054), the colorectal adenocarcinoma cell line SW620 (ATCC, CCL-227), and the ovarian adenocarcinoma cell line SKOV3 (ATCC, HTB-77) were kindly provided by the Department of Toxicology, Karolinska Institute (Stockholm, Sweden). Cells were routinely checked for mycoplasma. The cells were grown in Dulbecco’s modified Eagle medium (DMEM) with 4.5 g/L glucose (Gibco, Waltham, MA, USA) supplemented with antibiotic-antimycotic (Gibco), and 10% fetal bovine serum (Gibco). Cells were grown in a CO₂ incubator (5% CO₂) at 37 °C and re-cultured every 2–3 days using 0.15% trypsin solution (Gibco). Throughout the experiments, the cells at the logarithmic growth phase were treated with cisplatin (Teva, Tel Aviv, Israel) for 72 h. To block the apoptosis induced by this compound, a selective inhibitor of caspases and, subsequently, apoptosis Q-VD-Oph (Selleck Chemicals LLC, Houston, TX, USA), was added for 1 h prior to cisplatin.

Sazonova E.V., Yapryntseva M.A., Pervushin N.V., Tsvetcov R.I., Zhivotovsky B, & Kopeina G.S. (2024). Cancer Drug Resistance: Targeting Proliferation or Programmed Cell Death. Cells, 13(5), 388.

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Teratocarcinoma Cell Culture and Transfection

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The 2102Ep teratocarcinoma cells (kindly provided by Prof. Peter W. Andrews, University of Sheffield) [18 (link)] were grown in DMEM containing 4.5 g/L glucose (Gibco, Waltham, MA, USA) supplemented 10% fetal bovine serum (Gibco) and 2 mM GlutaMAX (Gibco, Waltham, MA, USA) in a 37 °C, 5% CO₂ atmosphere. Cultures were passaged using 0.25% trypsin/1mM EDTA. For experiments, cells were resuspended in fresh medium and seeded into fresh tissue culture flasks; 24 h before transfection, cells were seeded on six-well plates (2 × 10⁴ cells per well). Monolayers were approximately 70% confluent on transfection day (day ’0‘). Four hours before transfection, the culture medium was replaced with fresh DMEM without supplements. Cells were transfected with 3 µg of plasmid DNA using polyethyleneimine (Polysciences, Warrington, PA, USA). Briefly, plasmid DNA was diluted with buffer containing 0.15 M NaCl and 20 mM HEPES pH 7.5 and mixed with 15 µg of polyethyleneimine. The transfection mixture was incubated for 30 min at room temperature and then added to each well. After 24 h, the transfection medium was replaced with fresh supplemented medium. For stable transfection assays, antibiotic selection was initiated 48 h after transfection, using 0.2–1 µg/mL concentration of puromycin (Thermo Fisher Scientific, Waltham, MA, USA). Each experiment was performed at least three times.

Bereznicka A., Mikolajczyk K., Szymczak-Kulus K., Kapczynska K., Majorczyk E., Modlinska A., Piasecki T., Kaczmarek R, & Czerwinski M. (2021). Two Paralogous Gb3/CD77 Synthases in Birds Show Different Preferences for Their Glycoprotein and Glycosphingolipid Substrates. International Journal of Molecular Sciences, 22(18), 9761.

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