One million cells were plated onto 10 cm dishes in triplicate, grown overnight and treated with vehicle control (water) or with CQ or Q at the indicated doses for 3h at 37C. Cells were washed twice with PBS and the tissue culture media was replaced with media containing 0.8μCi of 1-14C-glucose (Perkin Elmer) or 6-14C-glucose containing vehicle (water) or the indicated antimalarial; cells were incubated for an additional 7h prior to analysis. To capture gaseous 14CO2, Whatman filter paper was taped to the inside of culture dish lid and the plate was then sealed with Parafilm; the experiment was terminated by adding 500μL of perchloric acid to the cells; the plate was resealed and left in the incubator overnight. Filter papers were then carefully removed and placed into vials containing Ready-Safe Liquid Scintillation Fluid (Beckman Coulter), and 14CO2 activity was measured on a Beckman LS6000 Liquid Scintillation Analyzer. The incubation of cells with 6-14C-glucose, used to correct for 14CO2 production from the citric acid cycle, yielded minimal 14CO2 activity.
6 14c glucose
Manufactured by PerkinElmer
Sourced in United States
[6-14C]-glucose is a radioactively labeled form of glucose, where the carbon atom at position 6 is labeled with the carbon-14 isotope. This product is commonly used as a tracer in various research and experimental applications to study glucose metabolism and associated biochemical processes.
Automatically generated - may contain errors
Lab products found in correlation
1 14c glucose, by PerkinElmer (6 mentions)
Ready safe liquid scintillation fluid, by Beckman Coulter (2 mentions)
Ls6000 liquid scintillation analyzer, by Beckman Coulter (2 mentions)
Hyamine hydroxide, by PerkinElmer (1 mentions)
Aconitase assay kit, by Cayman Chemical (1 mentions)
1 14c acetic acid, by PerkinElmer (1 mentions)
Citrate synthase assay kit, by Merck Group (1 mentions)
Succinate dehydrogenase activity colorimetric assay kit, by Abcam (1 mentions)
Isocitrate dehydrogenase activity assay kit, by Merck Group (1 mentions)
Alpha ketoglutarate assay kit, by Abcam (1 mentions)
14c l glutamine, by PerkinElmer (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Whatman filter paper, by Cytiva (1 mentions)
Whatman grade 1 filter paper, by Cytiva (1 mentions)
Cell culture grade, by Merck Group (1 mentions)
Uridine, by Merck Group (1 mentions)
Na2s nonahydrate, by Merck Group (1 mentions)
Monobromobimane fluoropure grade, by Thermo Fisher Scientific (1 mentions)
U 14c glutamine, by PerkinElmer (1 mentions)
11 protocols using 6 14c glucose
1
Glucose Oxidation Assay with Antimalarials
2
Measuring Glucose Oxidation via the PPP
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Glucose oxidation via the PPP was measured as previously described based on the difference in 14CO2 production from [1-14C] glucose (decarboxylated in the 6-phosphogluconate dehydrogenase-catalyzed reaction and in the Krebs cycle) and [6-14C] glucose (decarboxylated only in the Krebs cycle) (Cisternas et al., 2014 (link)). Slices were washed with ice-cold PBS and collected by trypsinization. The tissue was then resuspended in an O2-saturated Krebs Henseleit buffer, and 500 μl of this suspension (~106 cells) was placed in Erlenmeyer flasks with another 0.5 ml of Krebs Henseleit solution containing 0.5 μCi D-[1-14C] glucose or 2 μCi D-[6-14C] glucose and 5.5-mM D-glucose (final concentration). The Erlenmeyer flasks were equipped with a central well-containing an Eppendorf tube with 500 μl of benzethonium hydroxide. The flasks were flushed with O2 for 20 s, sealed with rubber caps, and incubated for 60 min in a 37°C water bath with shaking. The incubations were stopped by the addition of 0.2 ml of 1.75-M HClO4 into the main well, and shaking was continued for another 20 min to facilitate the trapping of 14CO2 by benzethonium hydroxide. Radioactivity was quantified as previously described by liquid scintillation spectrometry (Bolaños et al., 2008 (link); Herrero-Mendez et al., 2009 (link)). Both [1-14C] glucose and [6-14C] glucose were purchased from PerkinElmer (Waltham, MA, USA).
3
Glucose Metabolism Tracing via 14C Labeling
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After washing in PBS, cells were detached with PBS containing 2.5% v/v FBS and 2 mmol/L EDTA, rinsed with PBS, and resuspended in 1 mL Hepes buffer (145 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgSO4, 10 mmol/L Hepes, 10 mmol/L glucose, 1 mmol/L CaCl2, pH 7.4). 50 μL were taken up, sonicated and used to measure the protein content. In each remaining sample, 2 μCi of [6-14C] glucose (55 mCi/mmol, PerkinElmer) or 2 μCi of [1-14C] glucose (58 mCi/mmol, PerkinElmer) were added. The labelled cell suspension was incubated for 1 h in a closed tube to trap the 14CO2 produced from the [14C] glucose. After this incubation time, the metabolic flux was interrupted by adding 0.5 mL 0.8 N HClO4 [17 (link)]. [1-14C] glucose metabolized through PPP or TCA and [6-14C] glucose metabolized through the TCE only produced 14CO2. The amount of glucose producing CO2 through the PPP was obtained by subtracting the amount of [6-14C] glucose (TCA cycle) from [1-14C] glucose (TCA + PPP cycles), as described [17 (link)]. Results were expressed as nmol CO2/h/mg cell proteins.
4
Glucose Metabolism Quantification in Cells
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Cells were washed with fresh medium, detached with trypsin/EDTA (0.05/0.02% v/v), washed with PBS, and resuspended in 1 ml Hepes buffer (145 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgSO4, 10 mmol/L Hepes, 10 mmol/L glucose, 1 mmol/L CaCl2, pH 7.4) containing 2 μCi of [6-14C] glucose (55 mCi/mmol, PerkinElmer) or 2 μCi of [1-14C] glucose (58 mCi/mmol, PerkinElmer). A 50 μL aliquot was sonicated and used for the determination of the cell proteins. The remaining suspension of cells was incubated for 1 h in a closed experimental system to trap the 14CO2 produced from the [14C] glucose, and the reaction was stopped by injecting 0.5 mL 0.8 N HClO4, as described previously (42). 14CO2 is released when [1-14C] glucose is metabolized, either by the PPP or by the TCA, whereas it is only developed from [6-14C] glucose only via the TCA. The amount of glucose transformed into CO2 through the PPP was calculated as described [42 (link)] and expressed as nmol CO2/h/mg cell proteins.
5
Measuring Glucose Oxidation Flux in HDLECs
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Glucose oxidation flux was measured as previously described (53 (link)). Briefly, subconfluent HDLECs cultured in 12-well plates were incubated with 1 ml per well EBM2 medium (containing appropriate amounts of serum and supplement) with [6-14C]-glucose (PerkinElmer) for 6 h. Then, the cells were lysed using 12% perchloric acid, and the wells were covered immediately using filter papers soaked with hyamine hydroxide (PerkinElmer). After incubation in a fume hood for at least 12 h to reach saturation, filter papers were taken out, and the amount of evaporated 14CO2 was measured in a scintillation counter.
6
Tracing glucose flux through the TCA cycle
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Glucose flux through the TCA cycle was measured by radiolabelling 1 × 106 cells with 2 μCi [6-14C]-glucose (55 mCi/mmol; PerkinElmer, Waltham, MA, USA), [1-14C]-acetic acid (1 mCi/ml, PerkinElmer) or [14C]-L-glutamine (200 mCi/mmol, PerkinElmer). Cell suspensions were incubated for 1 h in a closed experimental system to trap the 14CO2 released from [14C]-glucose, [14C]-acetic acid], and [14C]-L-glutamine. The reaction was stopped by injecting 0.5 ml of 0.8 N HClO4. The amount of glucose transformed into CO2 through the TCA cycle was calculated as described in [103 (link)].
The enzymatic activity of citrate synthase, aconitase, IDH, alpha-ketoglutarate dehydrogenase, and SDH were measured on the basis of 10 µg of mitochondrial proteins using a citrate synthase assay kit (Sigma), an aconitase assay kit (Cayman Chemical, Ann Arbor, MI), an isocitrate dehydrogenase activity assay kit (Sigma), an alpha-ketoglutarate assay kit (Abcam), and a succinate dehydrogenase activity colorimetric assay kit (BioVision), as per the respective manufacturer’s instructions.
The enzymatic activity of citrate synthase, aconitase, IDH, alpha-ketoglutarate dehydrogenase, and SDH were measured on the basis of 10 µg of mitochondrial proteins using a citrate synthase assay kit (Sigma), an aconitase assay kit (Cayman Chemical, Ann Arbor, MI), an isocitrate dehydrogenase activity assay kit (Sigma), an alpha-ketoglutarate assay kit (Abcam), and a succinate dehydrogenase activity colorimetric assay kit (BioVision), as per the respective manufacturer’s instructions.
7
Glucose Metabolism in HT29 and HCEC Cells
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HT29 or HCEC (7 × 105 cells per well) were cultured in 12-well plates and incubated overnight under standard conditions described above for each cell line. Then, the medium was replaced (2 ml/well) with fresh RPMI 1640 medium containing Hepes buffer, supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin (Gibco) with 0.1 μCi per ml of [1-14C]- or [6-14C]-glucose (PerkinElmer) with or without Na2S (100 μM). The wells were covered with pieces of Whatman filter paper (grade 1) soaked with 1 M NaOH, and the cells were incubated at 37 °C in an ambient atmosphere. After 3 h, the cells were treated with a second bolus of Na2S (100 μM) and incubated for an additional hour. The reaction was terminated by addition of 300 μl of 3 M HClO4 per well, and the and the plates were kept overnight at ambient temperature. The Whatman filter papers were removed the next day, and radioactivity was measured using a liquid scintillation counter.
8
Mammalian Cell Culture Reagents
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Na2S nonahydrate (99.99% purity), glucose, hydrocortisone, insulin, apo-transferrin, sodium selenite, sodium orthovanadate, uridine (cell culture grade), protease inhibitor cocktail for mammalian tissue extract, puromycin (Sigma P8833), and RIPA buffer were from Sigma. Monobromobimane (FluoroPure grade) was from Molecular Probes. [1-14C]-glucose (56.5 mCi/mmol), [6-14C]-glucose (60.3 mCi/mmol), and [U-14C]-glutamine (281.0 mCi/mmol) were from PerkinElmer. Cell culture media (DMEM with 4.5 g/l glucose, glutamine, and 110 mg/l sodium pyruvate [Cat. # 11995], RPMI 1640 with glutamine [Cat. # 11875], 199 [Cat. # 11150]), fetal bovine serum (FBS) (Cat. # 10437), penicillin/streptomycin mixture (Cat. # 15140), PBS (Cat. # 10010), and DPBS (Cat. # 14040) were from Gibco.
9
Measuring Pentose Phosphate Pathway Activity
10
Glucose Oxidation Assay with Antimalarials
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