The medium was collected to detect glucose uptake, lactate production and pH. The glucose uptake and lactate production were assessed with glucose uptake assay kit and lactate assay kit (Sigma, USA) following manufacturer’s instructions. The oxygen consumption rate was measured using a Hansatech Oxytherm (Hansatech, King’s Lynn, UK).
Glucose uptake assay kit
Manufactured by Merck Group
Sourced in United States
The Glucose uptake assay kit is a laboratory tool used to measure the uptake of glucose by cells. It provides a quantitative assessment of glucose transport activity in cell-based assays.
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Lab products found in correlation
Lactate assay kit, by Merck Group (2 mentions)
Atp assay kit, by Merck Group (2 mentions)
L lactate assay kit, by Merck Group (2 mentions)
Oxytherm, by Hansatech (1 mentions)
Xf24 extracellular flux analyzer, by Agilent Technologies (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Rapamycin, by Thermo Fisher Scientific (1 mentions)
Metformin, by Thermo Fisher Scientific (1 mentions)
Isopropyl β d thiogalactopyranoside iptg, by Merck Group (1 mentions)
7 protocols using glucose uptake assay kit
1
Metabolic Profiling of Cell Cultures
2
Mitochondrial Function and Metabolism Assay
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Oxygen consumption rate (OCR) was determined using the XF24 Extracellular Flux Analyzer (Seahorse Bioscience; Agilent Technologies, North Billerica, MA) 19 . Glucose consumption, ATP content, and lactate production were evaluated using the Glucose Uptake Assay Kit, ATP Assay Kit, and Lactate Assay Kit (all from Sigma-Aldrich), respectively, according to the manufacturer's recommendations. To visualize mitochondria, cells were stained with MitoTracker Red CMXRos (Thermo Fisher Scientific) for 30 min. Nuclear counterstaining was performed using 4',6-diamidino-2-phenylindole (DAPI). Fluorescent images were acquired with a fluorescence microscope.
3
Antibody Staining and Signaling Assays
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Primary antibodies and Ig-matched controls (IgG1, IgG2b) were from BD Biosciences (Heidelberg, Germany), Thermo Fisher Scientific (Frankfurt, Germany), Santa Cruz Biotechnologies (Heidelberg, Germany) and Immunotools (Friesoythe, Germany). Isopropyl-β-D-thiogalactopyranoside (IPTG) and antibiotics were purchased from Sigma (Munich, Germany). The glucose uptake assay kit was also provided by Sigma. Leucine and arginine were purchased from Roth (Karlsuhe, Germany). Rapamycin, metformin and non-opsonized E. coli pHrodo particles were from Thermo Fisher Scientific. The antibodies to CD14 (clone MEM-18), phospho-mTOR (serine 2448, clone MRRBY), phospho-AKT (phospho serine 473 AKT1, 2, 3, rabbit polyclonal AF887), phospho-S6-Kinase (p-S6K, phospho-Thr 421, phosphor-Ser 424, clone E-5), phospho-S6 ribosomal protein (serine 235, serine 236, clone N7-548), PUMA (goat polyclonal IgG), LAT-1 (rabbit polyclonal IgG) phospho-4EBP (threonine 36, threonine 45, clone M31-16) BCL-XL (clone 7B2.5), BCL-2 (C-2), cleaved caspase-9 (clone D819E) and cytochrome C (clone 7H8.2C12) were used in dilutions recommended by the supplier.
4
Glucose, Lactate, and ATP Measurement
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Cells were cultivated into a 6-well plate. After transfection and/or treatment, supernatants of cell culture media were collected to detect the levels of glucose and lactate using the Glucose Uptake Assay Kit and l-Lactate Assay Kit (Sigma, St Louis, MO, USA) according to the protocol of the manufacturer. Glucose consumption and lactate production were calculated according to the percentage of the control group and normalized by the protein concentration of samples.
The ATP levels in cells were measured by an ATP Assay Kit (Sigma) according to the protocol of the manufacturer. Cells were lysed and then incubated with ATP reaction mix for 30 min. Finally, the optical density at 570 nm was measured by a microplate reader.
The ATP levels in cells were measured by an ATP Assay Kit (Sigma) according to the protocol of the manufacturer. Cells were lysed and then incubated with ATP reaction mix for 30 min. Finally, the optical density at 570 nm was measured by a microplate reader.
5
Glucose Uptake and Lactate Production
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After transfection and/or treatment, the supernatants of AC-16 cell culture media were collected, and subjected to the analysis of the consumption or production of glucose and lactate using a Glucose Uptake Assay Kit and L-Lactate Assay Kit (Sigma, St Louis, MO, USA) referring to the producer's guidance using a microplate reader. Experiments were performed three times.
6
Glucose Uptake Assay Protocol
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Glucose consumption was detected using a glucose uptake assay kit (Sigma-Aldrich) according to the manufacturer’s instructions. The cells were lysed using lysis buffer on ice. The samples (50 μL) were incubated with 8 μL assay buffer and 2 μL enzyme mix for 60 min at 37°C. Next, glutathione reductase (20 μL), substrate-DTNB (16 μL), and recycling mix (2 μL) were added in each well. The absorbance was measured at 412 nm.
7
Quantifying Glucose Uptake via SRSF10 Knockdown
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The BICR10 cells (3 × 105) were infected with lentivirus containing shRNA specific for SRSF10 gene in 6 well cell culture plates, and after 4 days glucose assay was performed according to the manufacturers instruction. Briefly, an equal number of cells were homogenized in the presence of glucose assay buffer provided in glucose uptake assay kit (Sigma, MAK083) and centrifuged at 13,000 g for 10 min. Glucose assay was then performed in a 96-well plate, and glucose levels was measured with a plate reader at an optical density of 570 nm.
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