HUVECs were purchased by the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultivated in EGM-2 Bullet Kit from Lonza (Walkersville, MD, USA). The cells were starved in medium containing with 0.25% albumin bovine serum (BSA) for 16 h and then respectively treated by low-glucose (5 mmol/L; Sigma, St Louis, USA), high-glucose (25 mmol/L; Sigma, St Louis, USA), and low-glucose combined with xylose (4.5 mmol/L; Sigma, St Louis, USA) for 24, 48, 72 h. In addition, different concentrations of MGO (0, 250, 500, 1000 μmol/L; Sigma, St Louis, USA) were added into HUVECs for 16 h, respectively. For comparison, cells were also pre-disposed with aminoguanidine bicarbonate salt (AG; 4 mmol/L; Sigma, St Louis, USA), and then treated by 500 μmol/L MGO for 16 h. Furthermore, insulin (100 nmol/L; Eli Lilly Florence, Italy) alone or in combined with MGO (500 μmol/L) were added into HUVECs for 10 min. For comparison, untreated cells (0 μmol/L) served as controls. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was performed in treated HUVECs to determine the expression level of miR-450a-5p.
Low glucose
Manufactured by Merck Group
Sourced in United States
The Low Glucose product is a laboratory equipment designed for the measurement and analysis of low glucose concentrations. It provides accurate and reliable results for research and clinical applications that require the detection of low levels of glucose in various sample types.
Automatically generated - may contain errors
Lab products found in correlation
High glucose, by Merck Group (3 mentions)
Pen strep, by Merck Group (2 mentions)
Mineral oil, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Huvecs, by Merck Group (1 mentions)
Hascs, by Lonza (1 mentions)
Egmtm 2 endothelial singlequots kit, by Lonza (1 mentions)
Hascs, by Merck Group (1 mentions)
Huvecs, by Lonza (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
Xylose, by Merck Group (1 mentions)
Egm 2 bulletkit, by Lonza (1 mentions)
Insulin, by Eli Lilly (1 mentions)
Lipofectamine and plus reagent, by Thermo Fisher Scientific (1 mentions)
D glucose, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Dmem hepes, by Thermo Fisher Scientific (1 mentions)
Rat tail collagen type 1, by Corning (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Pd98059, by Santa Cruz Biotechnology (1 mentions)
8 protocols using low glucose
1
Regulation of miR-450a-5p in HUVECs under Glucose and MGO Exposure
2
Tissue Engineering of Vascularized Bone Constructs
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In this study, human adipose stem cells (hASCs) (Lonza, USA) and human umbilical vein endothelial cells (HUVECs; Lonza) were used. The cells were cultured at 37 °C and 5% CO2 with different culture media, namely, growth medium (GM) for hASCs consisting of Dulbecco's Modified Eagle's Medium-low-glucose (DMEM-L; Sigma-Aldrich, USA), 10% fetal bovine serum (FBS; BioWest, USA), and 1% penicillin-streptomycin (PS; Thermo-Fisher Scientific, USA) and EBMTM-2 (Lonza) for HUVECs supplemented with the EGMTM-2 endothelial SingleQuotsTM kit (Lonza) and 1% PS (EBM). The culture medium was changed every 2 days.
Before formulating the stem cell-loaded bioink, a porcine bone-derived dECM (BdECM) sponge, prepared using a previously described demineralization/decellularization protocol (described inSupplementary materials ) 29 (link), was dissolved in deionized water and neutralized by mixing with 10×DMEM (Sigma-Aldrich) at a ratio of 1:1. The BdECM hydrogel was then mixed with b-TCP and hASCs (1.2 × 107 cells/mL). The final concentrations of dECM and- TCP were 50 and 200 mg/mL, respectively. To obtain cell spheroids, mineral oil (Sigma-Aldrich) containing HUVECs (2.0 × 107 cells/mL) was used. The total cell density to fabricate the cell-constructs was fixed as 2.0 × 107 cells/mL in which the ratio of the hASCs and ECs was 3:2.
Before formulating the stem cell-loaded bioink, a porcine bone-derived dECM (BdECM) sponge, prepared using a previously described demineralization/decellularization protocol (described in
3
Maintenance and Transfection of Human Cell Lines
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Human HCT116 cells (wild-type and p53-deficient mutant cells) [27 (link)] and HeLa cells were maintained in Dulbecco's modified MEM with high glucose and low glucose respectively (Sigma-Aldrich, St. Louis, MO USA) at 37 °C in the presence of 10% fetal bovine serum. Cell numbers were counted by the trypan blue dye-exclusion method with a hematocytometer. Etoposide dissolved in dimethylsulfoxide was added to the medium to 30–50 μm . Transfection of nucleic acids was performed by using Lipofectamine and Plus Reagent (Invitrogen, Carlsbad CA, USA).
4
Cardiac Fibroblast Response to Glucose and Thrombin
5
Cytotoxicity Evaluation of Nisin and N-BC
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The standard nisin and N–BC at the best time and with different concentrations of 7, 15, 31, 62, 125, 250 µg/mL were arranged in 24-well plates with 500 µL of DMEM (Dulbecco’s modified eagle’s medium, Low Glucose, Sigma®), with the addition of 10% fetal bovine serum (FBS, Sigma®) and 1% antibiotic (PEN/STREP, Sigma®) in each well. In each well, there were 75,000 L929 fibroblast cells. The plates were incubated at 37 °C in an atmosphere of 5% CO2 for 24 and 48 h. The assay was performed in triplicate and for positive control, PBS and DMEM were used.
After 24 and 48 h, cell viability was determined by the mitochondrial activity of the cell culture by the colorimetric assay MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]. Absorbance readings were taken at 490 nm, after 1 h of incubation of the cells with the reagent [27 (link),28 ]. The results are expressed as mean percentage ± standard deviation (SD) of viable cells in relation to the positive control.
After 24 and 48 h, cell viability was determined by the mitochondrial activity of the cell culture by the colorimetric assay MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]. Absorbance readings were taken at 490 nm, after 1 h of incubation of the cells with the reagent [27 (link),28 ]. The results are expressed as mean percentage ± standard deviation (SD) of viable cells in relation to the positive control.
6
Preparation of Collagen Gel for Cell Culture
7
TMAO Stimulation of HK-2 Cells
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HK-2 cells were stimulated with TMAO (300 µM, Sigma-Aldrich, St. Louis, MO, USA), low glucose (5 mM, unstimulated) or high glucose (30 mM, Sigma-Aldrich) for 3 min to 24 h, depending on the experimental setup, at 37 °C in 5% CO2. The HK-2 cells were also pre-incubated with DMSO (vehicle), Akt inhibitor MK-2206 (1 µM, Selleckchem, TX, USA), mTOR inhibitor ridaforolimus (1 µM, Selleckchem), PI3K inhibitor wortmannin (1 µM, Selleckchem) and ERK inhibitor PD98059 (10 µM, Santa Cruz Biotechnology Inc., Heidelberg, Germany) for 1 h prior to TMAO stimulation. Supernatants, cell lysates and total ribonucleic acid (RNA) were collected and kept at –80 °C until further analysis.
8
Scaffold Composition for Bone Tissue Engineering
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PEOT/PBT was provided by PolyVation BV (Groningen, The Netherlands). The commercial designation of this random block copolymer follows an aPEOTbPBTc nomenclature, where "a" is the molecular weight (Mw, g mol -1 ) of the poly(ethylene oxide) (PEO), "b" and "c" represent the weight ratios of PEOT and PBT, respectively. The block copolymer used in this study was 300PEOT55PBT45. Lowglucose (1000 mg L -1 ), Minimum Essential Medium Eagle -Alpha Modification (Alpha MEM), L-glutamine, penicillin-streptomycin (Pen-Strep), fungizone, trypsin, phosphate buffered saline (PBS), gelatin (Type B, 75 Bloom, from bovine skin), β-glycerophosphate, ascorbic acid, dexamethasone, Triton-X-100, neutral red, trypan blue, Mayer's hematoxylin solution, Eosin B, DPX mounting medium, absolute silver nitrate, pyrogallol, sodium thiosulfate, nuclear fast red, aluminum sulfate, ethanol, xylene, and methanol were provided by Sigma-Aldrich (Milan, Italy). Heat-inactivated fetal bovine serum (FBS), AlamarBlue® and paraffin histoplast LP from Thermo Fisher Scientific (Waltham, MA, USA). Lymphoprep was supplied by Axis-Shield, Norway. Calcium chloride for human use was purchased from Bioindustria Farmaceutici (Rome, Italy). EAC wall prosthesis TPL 07.39, was kindly supplied by Audiotechnologies (Gossolengo, PC, Italy). All the products were used as received, if not otherwise specified in the methods section.
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