Quantification of ecdysteroids in whole larvae was performed as described by [61 (link),113 (link)] with the following modifications. Briefly, animals were homogenized in 0.25 ml 75% methanol, and then the supernatants were collected following centrifugation at 14,000 g for 15 min. The pellets were re-extracted in 0.1 ml methanol. The supernatants were combined, evaporated using a SpeedVac, and then re-dissolved in 0.5 ml ELISA buffer (Cayman Chemical). Ecdysteroids were measured using a commercial ELISA kit (Cayman Chemical) that detects 20E equivalents. Standard curves were generated using 20E (Cayman Chemical), and absorbance was measured at 405 nm on a microplate photometer (Thermo Scientific).
Speed vac
Manufactured by Cayman Chemical
Sourced in United States
The Speed-Vac is a vacuum concentration system designed for the efficient removal of solvents from samples. It operates by creating a vacuum environment that facilitates the evaporation of liquid components, leaving behind the desired solid or semi-solid materials.
Automatically generated - may contain errors
Lab products found in correlation
Eia kit, by Cayman Chemical (2 mentions)
Elisa buffer, by Cayman Chemical (1 mentions)
Microplate photometer, by Thermo Fisher Scientific (1 mentions)
3 isobutyl 1 methylxanthine, by Enzo Life Sciences (1 mentions)
Cgmp eia buffer, by Cayman Chemical (1 mentions)
Imark automated plate reader, by Bio-Rad (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Mgcl2, by Thermo Fisher Scientific (1 mentions)
Tris hcl, by Thermo Fisher Scientific (1 mentions)
Sodium nitroprusside, by Merck Group (1 mentions)
Creatine phosphokinase, by Merck Group (1 mentions)
Creatine phosphate, by Merck Group (1 mentions)
Benchtop microplate reader, by Bio-Rad (1 mentions)
Eia buffer, by Cayman Chemical (1 mentions)
20 hydroxyecdysone enzyme immunoassay eia kit, by Cayman Chemical (1 mentions)
Vacuum manifold, by Waters Corporation (1 mentions)
Oasis hlb column, by Waters Corporation (1 mentions)
Sep pak c18 cartridge, by Waters Corporation (1 mentions)
5 protocols using speed vac
1
Ecdysteroid Quantification in Insect Larvae
2
Measuring sGC Activity via cGMP Assay
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30 μg of PA protein was measured using the Bradford method. Samples were incubated in a reaction mixture of 50 mM Tris-HCl (pH 7.5, Fisher Scientific), 4 mM MgCl2 (Fisher Scientific), 0.5 mM 3-isobutyl-1-methylxanthine (Enzo), 7.5 mM creatine phosphate (Sigma-Aldrich), 0.2 mg/mL creatine phosphokinase (Sigma-Aldrich), 1 mM sodium nitroprusside (Sigma-Aldrich), and 1 mM GTP (Sigma-Aldrich) at 37°C. After 10 minutes, the reaction was terminated using HCl (Sigma-Aldrich) to a final concentration of 0.1 N. Samples were dried using a Speed-Vac, and pellets were resuspended in 100–200 μL of cGMP EIA buffer (Cayman Chemical, Ann Arbor, MI). cGMP levels in the samples were measured in duplicate using a commercially available EIA kit (Cayman Chemical). Results were measured using a Bio-Rad iMark automated plate reader (Hercules, CA) at 405 nm [6 (link), 23 (link)]. sGC activity results are shown as pmol cGMP/minute/mg total protein.
3
Quantifying Larval Ecdysteroid Levels
4
Quantification of Lipid Mediators in BALF
5
Measurement of Hypothalamic PGE2 Levels
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The same procedures as previously published (Ayoub et al. 2004; Ayoub et al. 2006; Ayoub et al. 2008) were followed. Briefly, hypothalamic tissues or whole brains were pulverized with a nitrogen bomb (Biospec Products, Bartlesville, OK). One millilitre of 15% (v/v) ethanol (diluted in distilled water and then acidified to pH 3) was added to each tissue sample. The tissue homogenates were left at 4°C for 10 min and then spun at 375g for 10min at 4°C. C-18 Sep-Pak cartridges (Waters, Milford, MA, USA) were conditioned with 4ml of ethanol followed by 4ml of distilled water. The supernatant from tissue homogenates were then applied to the columns at a flow rate of 5 ml/min. The columns were then washed in 4ml of distilled water followed by 4ml of 15% ethanol in distilled water. The samples were finally eluted with 2 ml of ethyl acetate. The samples were dried in a Speed-Vac and then stored at -80°C.
Measurement of hypothalamic PGE2 was performed using a commercial enzyme immunoassay kit from Cayman Chemicals (Ann Arbor, MI, USA), according to the manufacturer's instructions. The concentration of PGE2 in the samples was determined by comparing the calculated percentage binding of PGE2 in the samples with a standard PGE2 curve (15.6-2,000pg/ml).
Measurement of hypothalamic PGE2 was performed using a commercial enzyme immunoassay kit from Cayman Chemicals (Ann Arbor, MI, USA), according to the manufacturer's instructions. The concentration of PGE2 in the samples was determined by comparing the calculated percentage binding of PGE2 in the samples with a standard PGE2 curve (15.6-2,000pg/ml).
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