Amyloglucosidase

Dissected FBs were homogenized in 300 µl ice-cold PBS using sonication followed by a syringe (29G1/2 needle). After reserving 20 µl of the homogenate for protein Bradford quantification, the rest of the homogenate was heat inactivated at 70 °C for 10 min. Homogenate was then centrifuged at maximum speed at 4 °C for 10 min and supernatant was collected in a new tube. In a 96-well plate, 30 µl of each sample was loaded in duplicate rows. Then, 100 µl of Autokit Glucose reagent +amyloglucosidase (1 µl amyloglucosidase {Sigma A1602; 25mg} per 1 ml of Glucose reagent) was added to one row of samples, and 100 µl of Glucose reagent alone (without amyloglucosidase) was added to the duplicate row of samples, and to the glucose standards. The plate was incubated at 37 °C for 30 min, after which color intensity was measured using a microplate reader at 505 nm. Free glucose concentration in treated and untreated samples was calculated based on the glucose standard curve. Glycogen concentration (as free glucose) was determined by subtracting glucose in the untreated samples from those treated with amyloglucosidase. Finally, total glycogen was normalized to protein measured by the Bradford assay.

Neutrophils (10 x10⁶) were cultured in U-¹³C pyruvate for 8 h under normoxia and hypoxia. The cells were washed with saline and lysed by boiling for 15 min in 30% KOH. Glycogen was precipitated using ice cold 100% ethanol and washed a further two times. Following the evaporation of residual ethanol and resuspension in water the glycogen was hydrolysed using amyloglucosidase (Merck Life Sciences UK). Acetonitrile:methanol:water 40:40:20 was added to the samples and samples analyzed by LC-MS.