Ecdysteroid Quantification in Insect Larvae

Fifteen larvae were rinsed with distilled water, and collected in a 1.5 ml microcentrifuge tube. The larvae were homogenized in 500 μl of methanol with a plastic pestle at room temperature. The samples were centrifuged at 15,000 g for 5 min at 4°C to obtain the supernatant. Ecdysteroid was quantitated by enzyme-linked immunosorbent assay (ELISA) using 20E EIA antiserum, 20E AchE tracer, and Ellman’s reagent (Cayman Chemical) as previously described [43 (link)].

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Ohhara Y., Kobayashi S, & Yamanaka N. (2017). Nutrient-Dependent Endocycling in Steroidogenic Tissue Dictates Timing of Metamorphosis in Drosophila melanogaster. PLoS Genetics, 13(1), e1006583.

Publication 2017

Ellman’s reagent

Ache Antiserum Cayman Ecdysteroid Ellman reagent Enzyme linked immunosorbent assay Larvae Methanol

Corresponding Organization : University of Tsukuba

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Variable analysis

independent variables

Rinsing with distilled water
Homogenization in 500 μl of methanol

dependent variables

Ecdysteroid quantitation by ELISA

control variables

Room temperature during homogenization
Centrifugation at 15,000 g for 5 min at 4°C

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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