Quantifying Ecdysteroid Levels in Drosophila

Flies were reared at 25°C and aged for 4 days. Ovaries were dissected in PBS. Samples were homogenized in 50 μL of methanol in 1.5-mL tubes using a pestle. After centrifugation at 20,913 x g for 1 min, the supernatants were transferred to new tubes and dried with a centrifugal evaporator. Samples were resuspended in 50 μL of EIA buffer (Cayman Chemicals) according to the manufacturer’s protocol and incubated at 4°C overnight. Ecdysteroid levels were quantified by an enzyme-linked immunosorbent assay using anti-20E antiserum (Cayman Chemicals) and 20E-acetylcholinesterase (Cayman Chemicals) as essentially described [62 (link)]. Note that the antiserum used in this study is known to recognize not only 20-hydroxyecdysone (20E) but also ecdysone [63 (link)]. In this study, we used we used 20E (ENZO Life Sciences) as a standard, and the ecdysteroid amount was expressed in 20E equivalents. Absorbance was measured at 415 nm using a microplate reader Multiskan GO (Thermo Fisher Scientific).

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Ameku T, & Niwa R. (2016). Mating-Induced Increase in Germline Stem Cells via the Neuroendocrine System in Female Drosophila. PLoS Genetics, 12(6), e1006123.

Publication 2016

EIA buffer 20E-acetylcholinesterase Multiskan GO

20 hydroxyecdysone Acetylcholinesterase Antiserum Buffer Cayman Centrifugation Ecdysone Ecdysteroid Enzyme linked immunosorbent assay Flies Methanol Ovaries

Corresponding Organization : Japan Science and Technology Agency

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Variable analysis

independent variables

Temperature: Flies were reared at 25°C

dependent variables

Ecdysteroid levels: Quantified by an enzyme-linked immunosorbent assay

control variables

Fly age: Flies were aged for 4 days
Dissection medium: Ovaries were dissected in PBS
Homogenization: Samples were homogenized in 50 μL of methanol
Centrifugation: Samples were centrifuged at 20,913 x g for 1 min
Resuspension: Samples were resuspended in 50 μL of EIA buffer and incubated at 4°C overnight

positive controls

20E-acetylcholinesterase (Cayman Chemicals) was used as a positive control

negative controls

No negative controls were explicitly mentioned in the protocol

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