Glucose go assay kit

Glucose concentrations in the harvested cell culture supernatants 72 h post-stimulation were determined using the Glucose (GO) Assay Kit (Sigma-Aldrich) according to [3 (link)]. The metabolic rate was calculated as the percentage of glucose consumption. For this, the measured glucose concentration in the samples was subtracted from the glucose concentration in the stock media (RPMI1640, 2 mg/mL). The Warburg Effect in culture media was determined as 1/OD570 nm, normalized to unstimulated controls according to [3 (link)].

Both glycogen and glucose were measured described elsewhere60 (link). For glycogen quantification, 3 mL sample was obtained and centrifuged; the pellet was collected and washed with sterile ddH₂O and then freeze dried. The weight of the dried cell was recorded. The cell was then suspended in 30% w/v KOH, mixed and incubated at 97 °C for 2 h. Ice-cold ethanol was added to the final concentration of 70% to 75% and incubated on ice for at least 2 h. The mixture was centrifuged, the supernatant was removed, and the pellet was washed with 98% ethanol. The glycogen pellet was dried at 60 °C for 10 min and then dissolved in 100 mM sodium acetate (pH 4.5). The dissolved glycogen was hydrolyzed enzymatically, by treating with 2 mg/mL amyloglucosidase (Sigma) at 60 °C for 2 h, into glucose. The glucose obtained from glycogen and extracellular (in media added externally) was measured using a Glucose (GO) Assay kit (Sigma-Aldrich) in accordance with the manufacture’s protocol. For glycogen measurement, data were normalized by cell dry weight. To understand the glucose uptake ability of the cell, kinetics of the decrease of the glucose content in the growth medium under GLC(+)DARK and GLC(+)DARK-LIGHT conditions were calculated. The data was normalized by OD₇₃₀ according to time duration.

To explore the effects of LsAkt RNAi on carbohydrate metabolism, the glucose, glycogen, and trehalose content assay was performed using a previously reported method (Xu et al., 2020 (link)). The contents of glucose, glycogen, and trehalose in whole insect bodies were measured by the SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA, United States) at 5 d after dsRNA treatment. Each sample contained 50 individuals, and three biological replications were prepared. The insect samples were homogenized in 0.25 M Na₂CO₃ and then incubated at 70°C for 10 min. By adding 0.2 M Na-acetate and 1 M acetic acid, the mixture was adjusted to pH 5.2. To measure the glycogen content, one half of the mixture was incubated with amyloglucosidase (Sigma-Aldrich). For trehalose measurement, the other half of the mixture was incubated with trehalase (Sigma-Aldrich) at 37°C, and the treated insects were homogenized in PBS (pH 7.4, 137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, and 2 mM KH₂PO₄₎. The glucose level was determined using a glucose (GO) assay kit (Sigma-Aldrich) according to the manufacturer’s instructions. After LsAkt was knocked down in L. serricorne, the transcript levels of eleven carbohydrate metabolic genes (Supplementary Table 1) were detected by qPCR at 5 d after injection.