Real time bioenergetics analysis of extracellular acidification rates (ECAR) and oxygen consumption rates (OCR) of T cells subjected to antibody stimulation was performed using the XF analyzer (Seahorse biosciences). T cells were cultured in serum free, unbuffered XF assay medium (Seahorse biosciences, Cat# 102365-100) for 1 hour. The cells were then seeded (6x105/well) into the seahorse XF24 cell plates for analysis. Perturbation profiling of the use of metabolic pathways by T cells was achieved by the addition of oligomycin (1 μM), FCCP (1 μM), Antimycin A (1 μM), rotenone (1 μM), D-glucose (10mM), 2-Deoxy-D-glucose (2DG, 50mM; all from Seahorse biosciences, Cat# 103020-100 and 103015-100). Experiments with the Seahorse system were done with the following assay conditions: 2 min mixture; 2 minutes wait; and 4–5 min measurement. Metabolic parameters were calculated. Experiments were done in at least triplicate wells.
2 deoxy d glucose 2 dg
Manufactured by Agilent Technologies
Sourced in United States
2-deoxy-d-glucose (2-DG) is a glucose analog that can be used as a tool compound in research applications. It serves as a substrate for glucose transporters and hexokinase, but is not fully metabolized, leading to disruption of cellular energy metabolism. The core function of 2-DG is to modulate glucose utilization in cells.
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Lab products found in correlation
Oligomycin, by Agilent Technologies (4 mentions)
Rotenone antimycin a, by Agilent Technologies (3 mentions)
Xf analyzer, by Agilent Technologies (1 mentions)
Rotenone, by Agilent Technologies (1 mentions)
D glucose, by Agilent Technologies (1 mentions)
Antimycin a, by Agilent Technologies (1 mentions)
Xf assay medium, by Agilent Technologies (1 mentions)
Nit 1, by RIKEN BioResource Center (1 mentions)
Antibody against β actin, by Merck Group (1 mentions)
Qgp 1, by Japanese Collection of Research Bioresources Cell Bank (1 mentions)
Rad001, by LC Laboratories (1 mentions)
F12 kaighn s medium, by Thermo Fisher Scientific (1 mentions)
Rapamycin, by LC Laboratories (1 mentions)
Cell tak, by Corning (1 mentions)
Seahorse xfe24 analyzer, by Agilent Technologies (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Xfe24 analyzer, by Agilent Technologies (1 mentions)
Fluoro carbonyl cyanide phenylhydrazone (fccp), by Agilent Technologies (1 mentions)
L glutamine, by Agilent Technologies (1 mentions)
Sodium pyruvate, by Agilent Technologies (1 mentions)
8 protocols using 2 deoxy d glucose 2 dg
1
Profiling T Cell Metabolic Signatures
2
Cell Line Characterization and Manipulation Protocols
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QGP-1, a human pNET cell line, was purchased from Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan). NIT-1, a mouse pNET cell line, was purchased from Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). The passage number of the two cell lines used in this study was less than 15. The QGP-1 cell line that we used was sent to the Center for Genomic Medicine of National Cheng Kung University for genotyping on Jun 2016, and the result showed the same STR DNA profile as those in the JCRB database. QGP-1 and NIT-1 cells were cultured in RPMI-1640 medium (Hyclone, South Logan, Utah, USA) and F12 Kaighn’s medium (Gibco, Grand Island, NY, USA), respectively, containing 10% fetal calf serum and antibiotics. The shRNAs targeting PCK2 and Pck2 were obtained from the National RNAi Core Facility of Academic Sinica (Taipei, Taiwan). Rapamycin and RAD001 were purchased from LC laboratories (Woburn, MA, USA) and Selleckchem (Munich, Germany), respectively. 2-Deoxy-D-glucose (2-DG) was purchased from Seahorse Bioscience (Billerica, MA, US). The primary antibody against PEPCK-M was purchased from GeneTex (GTX114919, USA). Antibodies against PEPCK-C (sc-74825) and GAPDH were purchased from Santa Cruz (Texas, US). Antibody against β-actin was purchased from Millipore (MA, US).
3
Glycolytic Rate Assay in E1A/RAS MEFs
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Sixty thousand E1A/RAS MEFs were plated in Seahorse 96-well cell culture microplates coated with Cell-Tak (Corning™) and subjected to the Seahorse XF Glycolytic Rate Assay, according to manufacturer’s protocol. Media was changed to Agilent Seahorse XF Base Medium without Phenol Red supplemented with 5.0 mM HEPES prior to the start of the assay. Measurements were performed after injection of three compounds affecting bioenergetics: 0.5 μM Rotenone/Antimycin A (Seahorse Bioscience), and 50 mM 2-deoxy-D-glucose (2-DG) (Seahorse Bioscience). Data are representative of three biological replicates.
4
Measuring Cellular Metabolism in HDFs
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The glycolysis rate and the oxygen consumption rate (OCR) in HDFs were measured with a Seahorse XFe24 analyzer (Agilent: Seahorse XFe24 analyzer, Santa Clara, CA, USA) and Wave Software (Agilent: wave controller, https://www.agilent.com/en/product/cell-analysis/real-time-cell-metabolic-analysis/xf-software/seahorse-wave-desktop-software-740897 , accessed on 5 March 2022). HDFs were seeded the previous day to reach 95% confluency before measuring. The glycolytic stress test measured the extracellular acidification rate (ECAR) following the manufacturer’s instructions. The OCR was measured using the Mito Stress test. DMEM media supplemented with 2 mM of L-glutamine (Thermofisher, Waltham, MA, USA) was used for the glycolytic stress test. A total of 10 mM of glucose (Agilent), 1 μM of oligomycin (Agilent), and 50 mM of 2-Deoxy-D-glucose (2-DG) (Agilent) were injected during the assay. For the MitoStress test, DMEM media supplemented with 10 mM of glucose, 1 mM of sodium pyruvate (Agilent), and 2 mM of L-glutamine were used. In total, 1.5 μM of oligomycin, 2 μM of FCCP (Agilent), and 0.5 μM of rotenone AA (Agilent) were injected during the assay.
5
Metabolic Profiling of Activated T Cells
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OCR and ECAR were determined using a Seahorse XF96 Extracellular Flux Analyzer (Seahorse Bioscience). The instrument was a gift from the S.D. Bechtel, Jr. Foundation to the Gladstone Institutes. Resting or 3-h anti-CD3–activated T cells (5 × 105 per well) were seeded onto Cell-Tak–coated wells (354240; Corning) in nonbuffered RPMI 1640 medium (R1383; Sigma-Aldrich), glucose depleted or supplemented with 11 mM glucose and 2 mM sodium pyruvate. Measurements were obtained under basal conditions and after adding 1 µM oligomycin, 0.5 µM FCCP, 0.5 µM rotenone/antimycin A (103015-100; Seahorse Bioscience), and 2-deoxy-d -glucose (2-DG) at a final well concentration of 50 mM (Agilent Technologies). Lactate measurements were detected using the primary metabolism screen conducted by the West Coast Metabolomics Center. In brief, sorted T cell populations were treated with 1 µM oligomycin and harvested at various times (0, 15, and 60 min). Samples were run on a mass spectrometer (ALEX-CIS GC-TOF; GERSTEL), and metabolites were quantified by peak heights and reported by retention index, quantification mass, biochemical database identifiers, and full mass spectra. Refer to Fiehn et al. (2008) (link) for further details regarding data acquisition and processing.
6
Seahorse Assay for Metabolic Phenotyping
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The Seahorse sensor probe plate was hydrated with XF calibrant 10 h before the test. The 24-well cell culture plates were coated with Poly-l -lysine (50 mg/ml, Solarbio, China) according to manufacturer’s instructions. 200,000 cells were plated in 200 ml XF Seahorse medium (pH 7.4, Agilent, USA) supplemented with 2 mM glutamine, 10 mM glucose and 1 mM sodium pyruvate. Plates were centrifuged at 200 g without break and cells were then incubated for 30 min at 37 ℃ without CO2. Afterwards, 300 ml of the same XF medium was added to each well. OCR was measured at basal conditions and after sequential stimulation of the cells by 1 mM Oligomycin (Oligo), 1 mM carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP) and 0.5 mM Rotenone/Antimycin (Rot/AA) (all included in the Mitostress kit, Agilent, USA). After the same pre-treatment, glycoPER was measured under basal conditions and with the sequential addition of 0.5 μM Rot/AA and 50 mM 2-deoxy-D-glucose (2-DG) (all included in the Glycolytic rate kit, Agilent, USA).
7
Metabolic Profiling of Hematopoietic Stem Cells
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The OCR and extracellular acidification rate were detected using the Agilent Seahorse XFp Cell Mito Stress Test Kit or the Glycolysis Stress Test Kit (Agilent Technologies, Santa Clara, CA, USA) as previously described (Hu et al., 2018 (link); Rao et al., 2019 (link)). Briefly, sorted LSKs (5 × 104) were plated into miniplates precoated with Cell-Tak (BD Biosciences). For the Mito Stress Test, cells were suspended in XF assay medium containing 1 mM pyruvate, 10 mM glucose, and 2 mM glutamine (pH 7.4), and then incubated in a CO2-free incubator at 37°C. Finally, respiration was measured by an Agilent Seahorse XFp analyzer (Agilent Technologies) after sequential addition of 1 μM oligomycin, 1 μM carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP), and 0.5 μM rotenone/antimycin A (Agilent Technologies). For the glycolysis stress test, cells were suspended in XF base medium supplemented with 1 mM glutamine (pH 7.4) and then incubated in a CO2-free incubator at 37°C. Finally, the glycolysis stress test was measured by an Agilent Seahorse XFp analyzer after the sequential addition of 10 mM glucose, 2 μM oligomycin, and 50 mM 2-deoxy-d -glucose (2-DG) (Agilent Technologies).
8
Cytokine Priming Enhances MSC Potency
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Based on a series of optimization experiments, CBti MSCs were primed with a regimen of four cytokines: interferon gamma (IFNγ) 10 ng/mL, interleukin 17 (IL-17) 10 ng/mL, interleukin 1 beta (IL-1β) 10 ng/mL, tumor necrosis factor alpha (TNFα) 10 ng/mL in alpha MEM media containing 1% of L-glutamine, for 16 h. For priming experiments, CBti MSCs were plated at high density (1.5 × 104 cells/cm2) and incubated overnight. The following day cells were pretreated with cytokines or left unprimed and incubated at 37°C and 5% CO2 for 16 h until sample collection. Supernatant was collected at this time for cytokine analysis. For experiments requiring blocking of glycolysis, MSCs were pretreated with 5 mM of 2 deoxy-D-glucose (2-DG) (Agilent). T cells were cultured in RPMI (Gibco) containing 10% fetal bovine serum (FBS), 1%L-glutamine, and 1% penicillin-streptomycin.
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