20e eia kit

Larvae staged at hatching as previously described were collected in three replicates of 10 animals for the later stages (84–112 h ALH) where the animals were larger and had more hormone to measure, and three replicates of 20 animals for the early stages (52–80 h ALH) when the larvae were very small. Results were calculated in pg per larvae to allow comparison. Larvae were collected from each population at 4 h intervals during L3. Larvae were washed in distilled water twice, briefly dried on paper towels and placed into ice cold methanol and kept at −80°C until use (Koyama et al., 2014 (link)). Prior to assaying, the samples were homogenized and centrifuged, the supernatant collected and the methanol from the supernatant was evaporated until completely dry (Mirth et al., 2005 (link)). Samples were resuspended in 100 μl enzyme immunoassay (EIA) buffer from the kit. EIA assay was performed as per the instructions of the 20E EIA kit purchased from Cayman Chemicals.

To estimate the titer of the ecdysteroids, 10 μL hemolymph was collected from the agomir-treated larvae at 24 and 48 h post-injection. A 20E EIA Kit (Cayman Chemical) was used to determine the 20E titer by following the manufacturers’ instructions. All experiments were conducted three times.

20E levels were quantified using the 20E EIA kit (Cayman Chemical). Nine females per time point were collected into the microtubes, snap frozen and kept at −80 °C. On the day of analysis, all samples were homogenized in 500 µl methanol using steal beads and a Qiagen tissue lyser at 50 rpm for 10 min. Samples were centrifuged at 15,000 rpm for 1 min and the supernatant (420 µl) was transferred to a clean tube. The methanol was evaporated in a speedvac. The pellets were dissolved in 230 µl of EIA buffer (Cayman Chemical). A 1:3 dilution series of 20E starting from 361 ng/ml served as a standard curve. Measurements were performed according to the manufacturer’s instructions.