As reported previously [35 (link)], the Glucose Colorimetric Assay Kit (Sigma‐Aldrich, MAK263) and Lactate Assay Kit (BioVision, Palo Alto, CA, USA, K607‐100) were used to determine glucose uptake and lactate production according to the manufacturer's protocols, respectively. Briefly, 3–5 × 105 PDAC cells were seeded into 6‐well culture plates. Twenty‐four hours later, cell culture supernatants were collected after 24 h and subjected for analysis. The final level of glucose and lactate was normalized to total protein content as measured by the Pierce BCA Protein assay (Pierce Biotechnology, Rockford, IL, USA).
Colorimetric glucose assay kit
Manufactured by Merck Group
Sourced in United States, Germany, Spain
The Colorimetric Glucose Assay Kit is a laboratory equipment product designed to measure glucose concentration in various samples. It utilizes a colorimetric method to quantify glucose levels accurately. The kit includes all the necessary reagents and instructions for performing the analysis.
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Lab products found in correlation
Lactate assay kit, by Abcam (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
Enzychrom l lactate assay kit, by BioAssay Systems (1 mentions)
Mak263, by Merck Group (1 mentions)
Bromocresol green, by Merck Group (1 mentions)
Milli q gradient system, by Merck Group (1 mentions)
Coomassie brilliant blue, by Merck Group (1 mentions)
Rhodamine b, by Merck Group (1 mentions)
Methyl orange, by Merck Group (1 mentions)
Milli q system, by Merck Group (1 mentions)
Vibratome, by Leica (1 mentions)
Phenol red, by Thermo Fisher Scientific (1 mentions)
10 protocols using colorimetric glucose assay kit
1
Glucose Uptake and Lactate Production Assay
2
Metabolic Effects of 64B in Tumor Cells
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Tumor cells (1.0 × 106 cells/well) were seeded in 6-well plates containing DMEM with 5.5 mM or 25 mM glucose. After 24 h, the medium was replaced with 2 mL fresh medium containing 64B (5 μM) and the cells were incubated for up to 24 h. Cells were lysed, and intracellular metabolites were measured using commercial kits detecting ATP (Abcam, Cambridge, MA), pyruvate (Sigma-Aldrich), acetyl-CoA (PicoProbe, Abcam) and NAD+/NADH ratio (BioVision, Milpitas, CA), respectively, according to the manufacturers’ recommended procedures. To measure pyruvate production, cells were cultured in pyruvate-free medium. Levels of glucose and lactate in the conditioned medium were analyzed by using a glucose colorimetric assay kit (Sigma-Aldrich) and an EnzyChrom™ L-Lactate assay kit (BioAssay Systems, Hayward, CA), respectively.
3
Glucose Quantification in Cell Supernatants
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The 24-h-simulated cell supernatants were collected by centrifugation (2,000 rpm, 1 min, RT) and then diluted 10 times with glucose assay buffer (catalog no. MAK263; Sigma, Germany). Ten microliters of the diluted supernatant was added to 40 μL glucose assay buffer to obtain 50 μL volume in a transparent 96-well plate for the glucose colorimetric assay kit (catalog no. MAK263; Sigma, Germany). The absorbance was measured at 570 nm with Synergy HTX multimode reader.
4
Glucose Metabolism in Schistosoma RNAi
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In a separate RNAi experiment, newly transformed schistosomula (5,000/treatment) were incubated for 5 days in SFB. Parasites were then electroporated with siRNAs as described above and finally transferred to serum-free DMEM (1 mg/ml glucose) supplemented with 4×AA. Media (50 μl) from each experiment was collected 48 h post-treatment and the amount of glucose was quantified using a colorimetric glucose assay kit (Sigma) following the manufacturer’s instructions. Parasite viability at this timepoint was determined by Trypan Blue exclusion and transcript levels of each smche, as well as the glucose transporters sgtp1 (smp_012440) and sgtp4 (smp_105410), were also measured. Glucose levels were normalized according to the number of viable parasites and expressed relative to the luc group. Data is the average of 2 biological and 3 technical replicates ± SEM.
5
Fabrication of Vertical Paper-Based Assay Devices
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Nitrocellulose (NC) was purchased from China Sinopham (Shanghai, China). Double-sided adhesive tape with different colours and tools for fabricating the vPADs were all bought from local grocery store and supermarket. The 5-bromo-4-chloro-3‘-indolyphosphate p-toluidine salt/nitro-blue tetrazolium chloride (BCIP/NBT) substrate solution and substrate buffer solution, sodium dodecyl sulfate (SDS) were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Coomassie brilliant blue, rhodamine B, methyl orange, bromocresol green, phosphate-buffered saline (PBS), polysorbate-20 (TWEEN-20), bovine serum albumin (BSA), Whatman grade 1 chromatography paper (20 cm × 20 cm) and colorimetric glucose assay kit was purchased from Sigma-Aldrich. The kits for colorimetric uric acid, cholesterol, triglyceride assays were purchased from Biosino Bio-Technology & Science Inc. (Beijing, China). Mouse anti-human myoglobin, human myoglobin, and alkaline phosphatase (ALP)-labelled goat anti-human myoglobin were purchased from Santa Cruz Biotechnology Co., Ltd (Shanghai, China). All solutions were prepared with deionised water (18.0 MΩ cm, Milli-Q Gradient System, Millipore) with ultraviolet sterilization. All reagents were used as received without further purification.
6
Glucose Metabolism in Primary Hepatocytes
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Primary hepatocytes were cultured for 8 h in the presence and absence of 1 μmol/L dexamethasone and 10 μmol/L forskolin in glucose‐ and serum‐free DMEM medium supplemented with 2 mmol/L sodium pyruvate and 20 mmol/L sodium lactate. In case of adenovirus‐infected primary hepatocytes, cells were cultured for 3 h in the same medium. The glucose concentration in the medium was measured using a colorimetric glucose assay kit (Sigma, St. Louis, MO) and normalized to the total protein content determined from the whole cell extracts.
7
Colorimetric Assays and Nanomaterials Synthesis
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Microcrystalline cellulose, the colorimetric protein assay kit was obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The colorimetric glucose assay kit was purchased from Sigma-Aldrich. Rhodamine B was purchased from Aladdin Bio-Chem Technology Co., Ltd. An oily slurry of conductive graphene was purchased from Nanjing XFNANO Materials Tech Co., Ltd. The monodisperse silica nanoparticles with diameters of 215, 225 and 288 nm were obtained from Nanjing Nanorainbow Biotechnology Co., Ltd. All reagents were of the best grade available and were used as received. All solutions were prepared with deionized water, which was prepared using a Millipore Milli-Q system (resistivity of 18.0 MΩ cm−1).
8
Liver Glucose Production Assay
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Glucose produc�on by precision cut liver slices (PCLS) derived from starved sham or CLP mice was measured 24 h a�er surgery. PCLS were derived from freshly isolated liver samples by slicing the big liver lobe with the vibratome (step size 250µm, amplitude 2mm, speed 0,1 mm/s; Leica). The slices are then punched with a 3 mm puncher and brought into a 24-well plate with 500µl of glucose-free DMEM, without phenol red, supplemented with 20 mM sodium lactate and 2 mM sodium pyruvate. A�er a 6-h incuba�on, medium was collected and the glucose concentra�on measured with a colorimetric glucose assay kit according to manufacturer's instruc�ons (Sigma). From each biological sample three slices were isolated as a technical repeat and mean value was used for further analysis.
9
Glycogen Metabolism in Parasite Cultures
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Five pairs of freshly perfused worms from each vaccinated group were cultured in DMEM (1000 mg/l glucose). Media (50 µl) from each experiment was collected after 24 h, and the amount of glucose was quantified using a colorimetric glucose assay kit (Sigma) according to the manufacturer's instructions. Glucose levels were expressed relative to media collected from worms recovered from PBS treated mice (negative control). To measure the glycogen content of these worms, Triton X-100-soluble extracts of each group of five pairs of worms (made by homogenizing the parasites in 1% Triton X-100, 40 mM Tris-HCl, pH 7.4, mixing overnight at 4°C and collecting the supernatant by centrifugation at 15,000 g for 1 h at 4°C) were assayed for glycogen in a modified procedure described by Gomez-Lechon et al. [16] (link). Briefly, 0.2 M sodium acetate, pH 4.8, was added to 30 μg parasite extract and 50 μl glucoamylase (10 U/ml) to make a reaction volume of 150 μl. The mixture was incubated at 40°C for 2 h with shaking at 100 rpm, 40 μl added to a new microplate with 10 μl 0.25 M NaOH and the amount of glucose quantified using the colorimetric glucose assay kit. Extracts were made from triplicate sets of parasites and assays were performed three times. Data are presented as the average of each triplicate biological and technical experiment SEM.
10
Glucose Production Quantification in NRK-52E Cells
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NRK-52E cells were seeded in 24-well plates (2 x 10 5 cells per well) and after the treatment, the medium was replaced with glucose production buffer consisting of glucose-free DMEM (pH 7.4) without phenol red (Invitrogen, Madrid, Spain), supplemented with 5 mM sodium lactate and 5 mM glutamine, as previously described [12, 21, 22] . After 1.5 h of incubation, medium was collected and the glucose concentration measured with a colorimetric glucose assay kit (Sigma, Madrid, Spain).
The readings were then normalized to the total protein content determined from the whole-cell lysates.
The readings were then normalized to the total protein content determined from the whole-cell lysates.
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