Glucose assay kit

A sample of coconut dregs collected from local market in Serdang, Selangor, Malaysia was ground and sieved into 1 mm particle size. It was separately mixed with different concentrations of sulphuric acid (0.2, 0.6 and 1%) and sodium hydroxide (1, 3, and 5%) at 50 °C for 3 days. Pre-treatment using different concentrations of cellulase (10,000 and 30,000 U/g of cellulysin (Merck, Malaysia) was done at 40 °C and pH 4 for 3 days. One unit of cellulose was measured for decomposing activity using a filter paper substrate at 40 °C and pH 4. At the end of the hydrolysis, liquid fraction from hydrolysate was filtered to be analysed for glucose concentration by glucose assay kit (Merck, Malaysia). The residual part was dried at 80 °C until a constant weight is achieved. The dried coconut dregs was kept in a dry state and used throughout the experiments.

3-mercaptopicolinic acid (3 MPA) treatment was performed at 1.5 mM concentration, metformin treatment at 250 μM concentration, and enzalutamide and bicalutamide treatments at 20 μM concentration. All other drug treatments were performed at 10 μM. For plate based screening, three 84 hpf ins mutant larvae were placed in each well of a 96-well plate in 200 μl of egg water buffered with 10 mM HEPES. All drug treatments were performed at 10 μM with 1% DMSO, unless otherwise stated. Drug treatment was performed from 84 to 120 hpf, after which each well was visually analyzed to assess toxicity. Subsequently, 100 μl of egg water was removed and 25 μl of 5X cell culture lysis buffer (Promega) was added. The plate was left shaking for 1 min at 750 rpm, and after gentle shaking at 150 rpm for 30 min, another round of vigorous shaking was performed for 1 min at 750 rpm. 8 μl from each well was used for the glucose detection reaction in a new 96-well plate using the Glucose Assay Kit (CBA086, Merck).
Drug libraries used in this screen are:

The glucose content in the medium was detected using a Glucose Assay kit (Merck, no. GAGO20) according to the manufacturer’s protocols. The rate of glucose consumption was calculated according to the standard curve line and OD value of each sample. Then, the values were also normalized by cell numbers determined by using a blood cell counting chamber.

Bacteria were grown in Gutnick minimal medium (33.8 mm KH₂PO₄, 77.5 mm K₂HPO₄, 5.74 mm K₂SO₄, 0.41 mm MgSO₄) supplemented with Ho-LE trace elements (54 ), 0.4% (w/v) glucose as the sole carbon source and NH₄Cl as the sole N source. Overnight cultures were grown at 37 °C, 180 rpm in Gutnick minimal medium containing 10 mm NH₄Cl. For the N starvation experiments, 3 mm NH₄Cl was used (see text for details). For carbon starvation experiments, bacteria were grown in Gutnick minimal medium containing 22 mm glucose and 10 mm NH₄Cl. The NH₄Cl and glucose concentrations in the media were determined using the Aquaquant ammonium quantification kit and glucose assay kit, respectively (both by Merck Millipore), according to the manufacturer's instructions. The proportion of viable cells in the bacterial population was determined by measuring CFU/ml from serial dilutions on lysogeny broth agar plates. Complementation experiments used pBAD24-hfq-3×FLAG or pBAD18 as the empty vector control, in Gutnick minimal medium supplemented with 0.2% (w/v) l-arabinose at t = 0, for induction of gene expression. To observe Hfq foci dissipation, 25 ml of N–24 culture was centrifuged at 3,200 g and resuspend in fresh Gutnick minimal medium containing different combinations of 0.4% (w/v) glucose and 3 mm NH₄Cl (see text for details).

Glycogen content was measured as described in by Cagin, U. et al. [75 (link)]. The glycogen was indirectly measured whilst the glucose was released after total digestion with amyloglucosidase from Aspergillus niger (Merck Life Science, Darmstadt, Germany). Sol8 myoblasts were differentiated into myotubes, as previously described. Lysates from cells and animal tissues were incubated for 5 min at 95 °C and then cooled at 4 °C; 25 μL of amyloglucosidase diluted at 1:5 in 0.1M potassium acetate (pH 5.5) was added to each sample. A control reaction without amyloglucosidase was prepared for each sample. Both sample and control reactions were incubated at 37 °C for 90 min. The reaction was stopped by incubating samples for 5 min at 95 °C. The released glucose was determined using a glucose assay kit (Merck Life Science) by measuring absorbance with Infinite 200 PRO NanoQuant (Tecan, Männedorf, Switzerland) at 540 nm.

The glycolytic ability of ccRCC cells was calculated using glucose uptake, lactate production and ATP analysis. Firstly, the glucose uptake level was measured using a glucose assay kit (Sigma, St-Louis, MO, USA) as previously described (Wang & Wang, 2022 (link)). Then, the lactate production level was detected by a lactate assay kit (Sigma, St-Louis, MO, USA). Lastly, the ATP analysis was performed using the Fluorometric Assay Kit (BioVision, Milpitas, CA, USA).

Glucose consumption was determined in culture media using a glucose assay kit (Sigma-Aldrich, St. Louis, MO, USA). Briefly, cell culture supernatants were harvested and diluted with deionized water, after which 50 μL of the diluted sample was mixed with an equal volume of assay buffer including o-dianisidine in a 96-well plate. The mixture was then incubated at 37 °C for 30 min, at which time the reaction was stopped by adding 50 μL of H₂SO₄ and the absorbance (O.D.) at 540 nm was measured in a microtiter reader (UVM340, Asys Hitech Gmbh, Eugendorf, Austria). The glucose consumption in each sample was calculated using a calculation formula from a standard curve.
Next, the cellular levels of glucose were measured in C2C12 myotubes using a glucose uptake cell-based assay kit (Cayman Chemical Co., Ann Arbor, MI, USA). Briefly, C2C12 myotubes were treated with or without yam extract and allantoin at different concentrations in glucose-free medium containing 100 µg/mL of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (NBDG) for 4 h. After harvesting the cells, cell-based assay buffer (200 µL) was added to each well. The amount of 2-NBDG taken up by the myotubes was then measured with fluorescent filters that detected fluorescein (excitation/emission = 485/650) using a Glomax multi detection system (Promega Biosystems, Sunnyvale, CA, USA).