Quantifying 20-Hydroxyecdysone in Drosophila

Female flies (25–30 flies/sample) were homogenized in 250 μL of ice-cold 100% methanol and centrifuged for 15 min at 18,000 g at 4 °C. Supernatants were transferred to 6 × 50 mm borosilicate glass tubes; precipitates were suspended in 200 μL of aqueous 75% methanol and kept on ice for 30 min (adapted from [63 (link)]). Samples were centrifuged and dried with a SpeedVac centrifuge. The precipitates were dissolved in EIA buffer at 4 °C and each individual sample was analyzed in a technical triplicate by competitive EIA using 20E EIA antiserum (Cayman Chemical: 482202) and 20E AChE Tracer (Cayman Chemical: 482200). Calibration curves were generated from commercial 20E (Sigma, H5142).

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Zheng W., Rus F., Hernandez A., Kang P., Goldman W., Silverman N, & Tatar M. (2018). Dehydration triggers ecdysone-mediated recognition-protein priming and elevated anti-bacterial immune responses in Drosophila Malpighian tubule renal cells. BMC Biology, 16, 60.

Publication 2018

20E EIA antiserum 20E AChE Tracer

Ache Antiserum Buffer Cayman Cold Female Flies Methanol

Corresponding Organization :

Other organizations : Brown University, University of Massachusetts Chan Medical School, University of North Carolina at Chapel Hill

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Levels of 20-hydroxyecdysone (20E)

control variables

Female flies (25–30 flies/sample)
Homogenization in 250 μL of ice-cold 100% methanol
Centrifugation for 15 min at 18,000 g at 4 °C
Precipitation and suspension in 200 μL of aqueous 75% methanol
Drying with a SpeedVac centrifuge
Dissolution of precipitates in EIA buffer at 4 °C
Technical triplicate analysis by competitive EIA using 20E EIA antiserum and 20E AChE Tracer
Calibration curves generated from commercial 20E

controls

Positive control: Commercial 20E (Sigma, H5142)
Negative control: Not explicitly mentioned

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