5 nM wild-type human GBA1 protein was incubated with varying amounts of glucosylceramide (Avanti Polor Lipids, #860547P) for various time points in reaction buffer containing Na2HPO4, 2.5 μg/ml Saposin C, 2.5 μg/ml Phosphatidylserine, 0.1% Triton X-100, adjusted to pH 5.0 with citric acid. At the end of each time point the reaction was terminated and the amount of glucose formed was determined using an equal amount the Amplex Red Glucose/Glucose Oxidase Assay Kit from Invitrogen (A22189) consisting of 100 μM Amplex Red reagent, 0.2 U/ml HRP, 2 U/ml glucose oxidase and 20 μM Isofagomine. For testing compounds, 0.75 nM glucocerebrosidase protein was incubated with 50 μM glucosylceramide (Avanti Polor Lipids, #860547P) for 30 minutes at room temperature in the same buffer, as described above. Glucose levels were determined using the Amplex Red Glucose/Glucose Oxidase Assay Kit from Invitrogen (A22189), as described above.
Amplex red glucose glucose oxidase assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Amplex Red Glucose/Glucose Oxidase Assay Kit is a fluorometric assay kit designed to detect and quantify glucose levels in samples. The kit utilizes the Amplex Red reagent, which reacts with hydrogen peroxide produced by the glucose oxidase enzyme, resulting in a fluorescent product that can be measured.
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114 protocols using amplex red glucose glucose oxidase assay kit
1
Glucocerebrosidase activity assay
2
Quantification of Cellular Glucose Uptake
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Cells were seeded in 24-well plates at 5~10 × 105 cells/well. Culture media were collected at 4 and 8h and stored at −20°C until they were assayed. Glucose uptake was measured using the Amplex Red Glucose/Glucose Oxidase Assay Kit (Molecular Probes, Carlsbad, CA, USA) or Glucose colorimetric/Fluorometric Assay Kit (BioVision, Mountain View, CA, USA). Absorbance was measured at 563 nm using a SpectraMax M5 plate reader (Molecular Devices, Sunnyvate, CA, USA) and results were normalized on the basis of the total protein amounts of the cells.
3
Glucose production measurement in Huh7 cells
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This was performed using Amplex Red Glucose/Glucose Oxidase Assay Kit (A22189, Molecular Probes, Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Briefly, Huh7 cells were transfected with pH19 or Vec in a 24-well plate. Forty-eight hours later, culture medium was replaced with glucose-free DMEM (Gibco; 11966-025) for 2 h and then incubated in 120 μl glucose production media (glucose-free DMEM, 20 mM sodium lactate, 2 mM sodium pyruvate, and 0.5% BSA) for 4 h. Subsequently, 50 μl of supernatant was used for measurement of glucose concentration, which was normalized to total protein content of cells.
4
Glycolysis Evaluation in Cells
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Treated cells were harvested and washed with PBS twice. To evaluate the glycolysis of cells, relative glucose consumption, lactate generation and LDH activity were detected by using Amplex Red Glucose/Glucose Oxidase assay kit (Molecular Probes; Thermo Fisher Scientific, Inc. USA), Lactate Assay Kit (BioVision, Milpitas, CA, USA) and LDH Quantification Kit (Biovision), respectively.
5
Glucose Uptake Measurement Protocol
6
Cancer Cell Metabolic Profiling
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After treatment with carboplatin (20 μM) and metformin (2 mM), A549/R and PC9/R cells were collected and washed with PBS for twice. Relative glucose uptake, lactate production and ATP production were detected by using Amplex Red Glucose/Glucose Oxidase assay kit (Molecular Probes, USA), Lactate Assay Kit (BioVision, USA) and ATP Colorimetric/Fluorometric Assay Kit (Biovision), respectively.
7
Glucose Uptake and Lactate Production
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Cells were seeded in 12-well plates at 1 × 105 to 3 × 105 cells per well. Culture media was collected at 48 hours and stored at −20 degree until assayed. Glucose uptake was measured using an Amplex Red Glucose/Glucose Oxidase assay kit (Molecular Probes). Absorbance was measured at 563 nm using a SpectraMax M5 plate reader (Molecular Devices) and the results were normalized to the amount of total protein compared with the control cells. Lactate production in the medium was detected by using a Lactate assay kit (BioVision). Results were normalized to the amount of total protein compared with the control cells.
8
Glucose Uptake and Lactate Production
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Cells were seeded onto 6-well plate, and then the pcDNA3.1-PKM2 (2 μg/ml), RNA oligonucleotides (NCO, miR-122 mimics, 50 nM) were transfected on the next day. After 24 h post-transfection, the cells were washed with PBS and incubated with fresh DMEM medium with 10% FBS in the absence or presence of DOX (10 μg/ml) for 24 h. The relative glucose uptake was measured using an Amplex Red Glucose/Glucose Oxidase assay kit (Molecular Probes, USA) according to the manufacturer’s protocol. Similarly, the lactate production in the medium was detected using a Lactate assay kit (BioVision, USA). The results were normalized to the NCO group.
9
Quantifying Hepatocyte Glucose Production
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Glucose production assays were performed using an Amplex Red Glucose/Glucose Oxidase Assay Kit (Molecular Probes, Invitrogen, A22189), according to the manufacturer’s instructions. Primary hepatocytes grown in 12-well plates were used. On the day of the assay, the culture medium was replaced with 500 μL of glucose-free and phenol red-free Dulbecco's Modified Eagle Medium (DMEM) (Gibco, A14430-01) supplemented with 2 mM L-glutamine and 15 mM Hepes for 2 h. Then, cells were incubated in 240 μL of glucose production medium (glucose-free and phenol red-free DMEM, supplemented with 20 mM of sodium lactate, 2 mM of sodium pyruvate, 0.5% bovine serum albumin, 2 mM of L-glutamine, and 15 mM of Hepes) for 4 h. Supernatants (200 μL) were used for measurements of glucose concentration, which was normalized to total protein content of cells.
10
Glucose Production Assay in Hepatocytes
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Glucose production assays were performed using Amplex Red Glucose/Glucose Oxidase Assay Kit (Molecular Probes, Invitrogen, A22189), according to the manufacturer’s instructions. Briefly, primary hepatocytes grown in 12-well plates were used. On the day of the assay, CM was replaced with glucose-free and phenol red-free DMEM (Gibco, A14430-01) supplemented with 2 mM l -glutamine and 15 mM HEPES for 2 h. Then, cells were incubated in 120 μl of glucose production medium (glucose-free and phenol red-free DMEM, 20 mM sodium lactate, 2 mM sodium pyruvate, and 0.5% BSA, 2 mM l -glutamine, and 15 mM HEPES) for 4 h. Supernatants (50 μl) were used for measurements of glucose concentration, which was normalized to total protein content of cells.
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