Np40 substitute assay reagent

Hemolymph JH III levels from WT and KO female Ae. aeygpti mosquitoes was quantified using previously described procedures with slight modifications. Briefly, hemolymph (5 μL) from five individuals was collected by perfusion as described above, with each pool constituting a single biological replicate. A 10 μl aliquot of deuterated JHIII analog (6.25 ppb) in acetonitrile was added to each sample before extraction with 600 μl hexane. The organic phase was dried under a stream of nitrogen and resuspended in 50 μl of acetonitrile and subjected to analysis by HPLC-MS/MS analyses [39 (link)].
Total triglycerides were quantified using the Triglyceride Colorimetric Assay Kit (Cayman Chemical) following the manufacturer’s instruction. Briefly, WT and KO Ae. aegypti female mosquitoes were collected at 6, 12, 24, 48, 72, 96 and 120 h after adult emergence. Pools of five mosquitoes at each time point were homogenized in 100 μl NP40 Substitute Assay Reagent (Cayman Chemical). The sample solution (10 μl) was incubated with 150 μl of diluted Enzyme Mixture solution (Cayman Chemical) and the absorbance (540 nm) was measured using a Microplate Reader.

Hepatic triglyceride content was measured using a triglyceride colorimetric assay kit (Cayman Chemicals, Ann Arbor, MI, USA) according to the manufacturer’s instructions. Briefly, liver tissues (40 mg) were homogenized in 200 µL of the NP40 substitute assay reagent (Cayman Chemicals) containing protease inhibitors (Thermo Fisher Scientific, Waltham, MA, USA), and centrifuged for 10 min at 10,000× g at 4 °C to collect the supernatants. The sample and enzyme mixture were incubated for 30 min at 37 °C and the absorbance was measured at 540 nm using a VersaMax microplate reader (Molecular devices, San Jose, CA, USA).