Respiratory capacity and glycolytic activity of cells were performed with the Seahorse XF24e Extracellular Flux Analyser (Seahorse Bioscience, Billerica, MA, USA) on attached cells as described9 (link). Briefly, 2 × 104 cells/well were seeded in XF24 V7 microplates for 24 h before drug treatment. To determine oxygen consumption rate (OCR), cells were resuspended in DMEM (D5030, Sigma-Aldrich) with l -glutamine (2 mM) and d -glucose (10 mM) and the following drugs were added: 1 µM oligomycin, 0.25–0.5 µM FCCP, 1 µM rotenone, and 1 µM antimycin A. To determine extracellular acidification rate (ECAR), cells were resuspended in glucose-free DMEM (D5030, Sigma-Aldrich) with l -glutamine (2 mM) and the following compounds were added: d -glucose (10 mM), oligomycin (1 µM), and 2DG (10 mM).
D5030
Manufactured by Merck Group
Sourced in United States
The D5030 is a laboratory equipment designed for general laboratory use. It is a versatile device that can be utilized for various experimental and analytical applications. The core function of the D5030 is to provide a reliable and consistent platform for performing laboratory tasks. No further details or interpretation on its intended use are provided.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Merck Group (5 mentions)
Seahorse xfe96 analyzer, by Agilent Technologies (4 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
F2442, by Merck Group (3 mentions)
Seahorse xf96 cell culture plate, by Agilent Technologies (3 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Seahorse xf imaging and normalization system, by Agilent Technologies (2 mentions)
13c glucose, by Cambridge Isotopes (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Biopsy puncher, by Merck Group (2 mentions)
Islet capture microplates, by Merck Group (2 mentions)
Seahorse xf cell mito stress test kit, by Agilent Technologies (2 mentions)
Wave software, by Agilent Technologies (2 mentions)
Seahorse xf24 extracellular flux analyzer, by Agilent Technologies (2 mentions)
Reduced glutathione, by Merck Group (2 mentions)
Rapamycin, by Merck Group (2 mentions)
83 protocols using d5030
1
Cellular Metabolic Profiling using Seahorse
2
Measuring Mitochondrial Function in Brown Adipocytes
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Oxygen consumption rate (OCR) was measured using the Seahorse XFe96 Analyzer (Agilent) on day 8 of differentiation. One hour prior to respirometry, media was replaced with basal DMEM (Sigma Aldrich D5030) supplemented with 5 mM glucose, 2 mM GlutaMax TM (Fisher Scientific 13462629) and 2% fatty acid-free BSA (Sigma Aldrich A3803), and cells were incubated at 37°C in a non-CO2 incubator. NE (final concentration 2 μM) was injected to stimulate brown adipocytes. ATP-linked respiration and uncoupling were determined by injecting Oligomycin (final concentration 5 μM) and Antimycin A (final concentration 5 μM) respectively. Basal mitochondrial respiration in stimulated cells was determined by subtracting the non-mitochondrial respiration from NE-induced OCR. Raw data were normalized by in situ nuclear staining (Hoechst 33342, final concentration 10 μM) and in situ cell counting using Seahorse XF Imaging and Normalization system (Agilent). Normalized data were used for calculations.
3
Campylobacter Growth Under Anaerobic Conditions
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Campylobacter jejuni precultures were grown for 7 h in HI medium with the addition of 50 mM nitrate at oxygen limiting conditions at 42°C. The precultures were diluted 50 times in 300 μL DMEM (without glucose, glutamine, pyruvate, bicarbonate, and phenol red, D5030, Sigma) with the addition of 10 mM TMAO and 10 mM glutamate, glutamine or aspartate, and growth curves were generated at 42°C in a 100 well honeycomb plate which was continuously shaking in a Bioscreen C MRB (Oy Growth Curves Ab) computer-controlled incubator. The incubator was placed inside an anaerobic chamber (Coy Labs, Grass Lake, MI, USA), due to suboptimal gas exchange in the honeycomb plate the oxygen concentration was set to 1%, which yielded comparable growth as when bacteria were grown in rectangular flasks inside an anaerobic jar containing 0.3% O2. The OD600nm of cultures was recorded every 15 min during 45 h. For clarity reasons only point at 2.5 h, or 5 h intervals are shown. Experiments were repeated three times in duplicate.
4
Metabolic Analysis of SC-β Clusters
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SC-β clusters were enriched using TSQ-based sorting and reaggregation. After reaggregation cells were washed and incubated in simple DMEM medium (D5030, Sigma-Aldrich) created from powder on the day of analysis using 4 mM Glutamine and 9 mM Glucose as previously described (Alves et al., 2015 (link)). After allowing clusters to reach steady state metabolism for 3 hours, clusters were treated with uniformly labeled 13C glucose (Cambridge Isotope Laboratories) for 0-180 minutes followed by lysis in quench buffer described by Alves et al. After quenching, samples were frozen on dry ice and shipped overnight to Yale University for Mass Spectrometric analysis. Analysis was carried out using electrospray into an ABSCIEX 5500 QTRAP with SelexION differential mobility separation using the same settings in multiple reaction monitoring in negative mode as previously described (Alves et al., 2015 (link)). Values were generated as a function of signal enrichment and described at Atomic Percent Enrichment (APE). Final values were calculated at Yale University by TA using Wave software and using the same standards for labeled intermediates as described in Alves et al. (2015) (link).
5
BM-MSCs Osteogenic Differentiation and Autophagy
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Human bone marrow mesenchymal stem cells (BM-MSCs; catalog no. PCS-500-012) were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM; D5030, Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; F2442; Sigma-Aldrich, St Louis, MO, USA), 100 U/mL Penicillin (Invitrogen, USA), and 100 μg/mL Streptomycin (Invitrogen, USA).
For osteogenic differentiation, the BM-MSCs were cultured in DMEM (Invitrogen, USA) containing 1% FBS (F2442; Sigma-Aldrich, USA), 200 μMl -glutamine (G7513; Sigma-Aldrich, USA), 10 nM dihydroxyvitamin D3 (D1530; Sigma-Aldrich, USA), 10 mM β-glycerolphosphate (50020; Sigma-Aldrich, USA), 100 nM dexamethasone (D4902; Sigma-Aldrich, USA), and 80 μg/mL ascorbic acid phosphate (A8960; Sigma-Aldrich, USA). And the medium was refreshed every 3–4 days.
To evaluate the effects of Forkhead Box O3 (FOXO3) on the autophagy of BM-MSCs, 100 nM autophagy activator Rapamycin (RAPA; R0395; Sigma-Aldrich, USA) was added into the BM-MSCs transfected with lentivirus carrier of small interfering RNA for FOXO3 (siFOXO3). 5 mmol/L autophagy inhibitor 3-methyladenine (3-MA; M9281; Sigma-Aldrich, USA) was added into BM-MSCs transfected with lentivirus carrier of overexpressed FOXO3 plasmid.
For osteogenic differentiation, the BM-MSCs were cultured in DMEM (Invitrogen, USA) containing 1% FBS (F2442; Sigma-Aldrich, USA), 200 μM
To evaluate the effects of Forkhead Box O3 (FOXO3) on the autophagy of BM-MSCs, 100 nM autophagy activator Rapamycin (RAPA; R0395; Sigma-Aldrich, USA) was added into the BM-MSCs transfected with lentivirus carrier of small interfering RNA for FOXO3 (siFOXO3). 5 mmol/L autophagy inhibitor 3-methyladenine (3-MA; M9281; Sigma-Aldrich, USA) was added into BM-MSCs transfected with lentivirus carrier of overexpressed FOXO3 plasmid.
6
Extracellular Flux Analysis of Cellular Metabolism
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Experiments were performed as in (17 (link)). In brief, XF96 extracellular flux analyser (Seahorse Bioscience; XF96 FluxPak Agilent Technologies) was used for OCR and ECAR measurements. Glucose-based mitostress test was measured in human primary T lymphoblasts or Jurkat E6-1 cells cultured with DMEM medium (D5030, Sigma Aldrich) supplemented with 2 mM sodium pyruvate, 2 mM L-glutamine and 25 mM glucose and drugs injected as follows: oligomycin (2 µM), CCCP (2.5 µM), rotenone plus antimycin A (1.5 µM each). For glycolysis stress test, cells were cultured with DMEM medium supplemented with 3.5 mM L-glutamine. Glucose (10 mM) was injected, followed by oligomycin (1.5 µM) and 2-deoxyglucose (2-DG, 50 mM). A linear mixed model performed in R v4.2.1 (https://cran.r-project.org ) was used for statistical analysis (17 (link)).
7
NIT-1 Cell Line Cultivation Protocol
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An NIT-1 cell line was purchased from Taiwan Bioresource Collection and Research Center (BCRC number: 60452). NIT-1 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (D5030, Sigma-Aldrich) supplemented with 16.5 mM glucose, 2 mM L-glutamine, 15 mM HEPES, 0.02% BSA, 1% penicillin-streptomycin (15140122, Gibco), and 10% heat-inactivated (56 °C for 30 min) and dialyzed fetal bovine serum (10735086001, Roche). Cells were maintained at 37 °C with 5% CO2 in a humidified incubator. The NIT-1 cells used in this article were all from passage 38 to passage 47.
8
Adipose Tissue Metabolic Profiling
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Subcutaneous white adipose tissue (1.5-2 mg) was obtained using a Biopsy Puncher (2 mm) and placed into XF24 Islet Capture Microplates and incubated in assay medium (substrate-free DMEM (Sigma D5030), 30 mM NaCl, pH7.4, 25 mM glucose, 0.5 mM sodium pyruvate). Oxygen consumption rate was measured on a Sehaorse XF24 Flux analyser following sequential addition of oligomycin (5 μM), carbonyl cyanide 3-chlorophenyl hydrazone (CCCP, 5 μM) and antimycin (5 μM).
9
Evaluating Phenformin Oxygen Consumption
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The cells were seeded at 15k in multiple XFp cell culture plates and left overnight to attach prior to conducting the experiment. The following day, medium was removed and cells were washed with PBS before addition of unbuffered serum-free, phenol red-free DMEM (D5030, Sigma-Aldrich, St. Louis, USA). The DMEM medium was adjusted to pH 7.4 and supplemented with 2 mM l -glutamine (Corning, New York, USA) and 5 mM glucose (Sigma-Aldrich, St. Louis, USA), and the cells were left for one hour to equilibrate at 37 °C in a CO2-free incubator. The measurements included three basal oxygen readings (5 minutes apart) before addition of 100 μM free phenformin or phenformin loaded to GO or PGNSs to initiate an acute response. The decrease in oxygen consumption was monitored over 75 minutes before increasing the total concentration to 600 μM phenformin, with sampling of data every 20 minutes until completion of the experiment (250 minutes).
10
Glucose Metabolism Profiling in Jurkat Cells
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The use of glucose was measured in siCtrl or siCCT Jurkat E6‐1 cells cultured with DMEM medium (D5030, Sigma Aldrich) supplemented with 1 mM sodium pyruvate, 1 mM L‐glutamine and 25 mM glucose. Cells were seeded at 0.3 × 106 per well in poly‐L‐Lys pre‐coated culture plates. Each plate included five biological replicates and four technical replicates. Drugs were injected as follows: oligomycin (1 μM), CCCP (1.5 μM), rotenone (1 μM) plus antimycin A (1 μM). Three consecutive mix and measure steps were performed for resting conditions and after each injection (3 min each).
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