Water → (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal

At 72 hpf, larvae were placed into a 96-well plate (6 eggs/well in 200 μL E3 water supplemented with 0.2 mM 2-phenylthiourea; Sigma). Drug of interest was added for h. The solution was then replaced with E3 water supplemented with 600 μM 2-NBDG and incubated for 3 h. The larva were washed with E3 water and anesthetized with 0.02% tricaine-supplemented E3 water. One larva was then placed on a chamber slide, containing 3% methylcellulose in E3 water, for fluorescent microscopy (Leica DM2500 microscope equipped with a DFC425 C digital camera). Images were captured at 50× magnification. The remaining 5 larvae where lysed with 120 μL of CelLytic M solution (Sigma-Aldrich) and sonication (4° C., 10″/5″ pulse, 10 min). Lysed larvae were centrifuged at 10000 rpm for 10 min. 100 μL of the supernatant was transferred to a 96-well plate and fluorescence was measured with a fluorescent microplate reader (SpectraMAX Gemini XS, Molecular Devices; λex=466 nm λex=540 nm).

1. additionDrug of interest was added for h
2. washThe larva were washed with E3 water
3. additioncontaining 3% methylcellulose in E3 water
4. customfor fluorescent microscopy (Leica DM2500 microscope equipped with a DFC425 C digital camera)
5. customThe remaining 5 larvae where lysed with 120 μL of CelLytic M solution (Sigma-Aldrich) and sonication (4° C., 10″/5″ pulse, 10 min)
6. waitLysed larvae were centrifuged at 10000 rpm for 10 min

ORD record

ord-8674d7595aa9455a948849070f04aad5

Source dataset

uspto-grants-2016_06

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US09364480B2