The wild type N2 C. elegans was provided by the Caenorhabditis Genetics Center (CGC). Resveratrol and FUDR (Sigma Aldrich) were used as the test compounds. Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a type of natural phenol with low aqueous solubility and the molecular weight of 228.24 (Figure 1A ). Resveratrol has been shown to extend the lifespan of yeast, fly and C. elegans with clear mechanism of action [30] (link)–[33] (link). FUDR, with the molecular weight of 246.2 (Figure 1B ), was soluble in water at the concentration to 50 mg/mL. FUDR was widely used to inhibit the worms to lay eggs in research with the concentration of FUDR from 40 µM to 50 µM [34] , [35] (link).
Resveratrol was dissolved in DMSO to 100 mM as stock solution. In LB medium method, the resveratrol stock solution was diluted into LB liquid medium containing OP50 E. coli bacteria to a final concentration of 100 µM and 1.5 mL of the bacteria solution was applied to each NGM plate (100 mm diameter). In spot dead method, 1.5 mL of 100 µM resveratrol was spotted onto the surface of the NGM plate, then covered with dead bacteria. In NGM live method, the resveratrol stock solution was diluted with NGM (below 65°C after boiled) to the concentration of 100 µM. Then the NGM was poured into petri plates as supporting bed for worms and live OP50 was applied to the surface of NGM plates. In NGM dead method, the plates were made as the NGM live method, except that the food OP50 bacteria was killed by incubating in 65°C for 30 minutes [24] (link). The drug administration of above four methods was summarized inFigure 2 . The maintenance of C. elegans in liquid medium was described as previously [23] with slight modification. Briefly, the synchronized N2 adult day 1 worms were cultured in 50 mL centrifuge tubes that contained 35 mL S medium [23] , the concentration of resveratrol in S medium was 100 µM. Dead bacteria were added to S medium as food. FUDR was dissolved in H2O to 50 mM as stock solution. The procedures of treatment of worms with FUDR were the same with resveratrol, except that the final concentration of FUDR in the five treatment methods was 50 µM.
About 5,000–10,000 adult day 1 wild type worms were transferred to each NGM plate (100 mm diameter). For the liquid growing method, the worms were cultured in several 50 mL centrifuge tubes with each containing about 35 mL S medium [23] . Worms in each method were harvested by using cold M9 buffer at the 10 min, 30 min, 1 hr, 3 hr, 6 hr, 12 hr, day 1, day 2, day 4, day 7, day 14 and day 20 after treated with the compounds [23] , and collected to 15 mL centrifuge tubes. The control group without treatment with compound was also harvested. The tubes were putted into ice for 10 minutes, then spin for 2 minutes at 1,150× g to precipitate the worms. The worm pellets were rinsed three times with cold M9 buffer, air dry, and weighed. The worm pellets were resuspended by using 1 mL methyl ethanol (HPLC grade) (for resveratrol) or H2O (for FUDR) and sonicated 50 times (200 V, operation 5 seconds every 5 seconds). Then, the worm solution was centrifuged under 12, 000× g for 3 minutes. The supernatant of the worm solution containing resveratrol or FUDR was transferred to a 1.5 mL centrifuge tube.
To test the drug metabolism, the worms treated with 400 µM, 200 µM, 100 µM, 50 µM, 25 µM, and 12.5 µM of resveratrol and FUDR, respectively, for 6 hours under NGM dead method were transferred to NGM plates without resveratrol and FUDR. Then, the worms were harvested at the 10 min, 30 min, 1 hr, 2 hr, 3 hr, 4 hr, 6 hr, 8 hr, 12 hr, and 16 hr time points. Subsequent sample preparation was the same as described above.
Resveratrol was dissolved in DMSO to 100 mM as stock solution. In LB medium method, the resveratrol stock solution was diluted into LB liquid medium containing OP50 E. coli bacteria to a final concentration of 100 µM and 1.5 mL of the bacteria solution was applied to each NGM plate (100 mm diameter). In spot dead method, 1.5 mL of 100 µM resveratrol was spotted onto the surface of the NGM plate, then covered with dead bacteria. In NGM live method, the resveratrol stock solution was diluted with NGM (below 65°C after boiled) to the concentration of 100 µM. Then the NGM was poured into petri plates as supporting bed for worms and live OP50 was applied to the surface of NGM plates. In NGM dead method, the plates were made as the NGM live method, except that the food OP50 bacteria was killed by incubating in 65°C for 30 minutes [24] (link). The drug administration of above four methods was summarized in
About 5,000–10,000 adult day 1 wild type worms were transferred to each NGM plate (100 mm diameter). For the liquid growing method, the worms were cultured in several 50 mL centrifuge tubes with each containing about 35 mL S medium [23] . Worms in each method were harvested by using cold M9 buffer at the 10 min, 30 min, 1 hr, 3 hr, 6 hr, 12 hr, day 1, day 2, day 4, day 7, day 14 and day 20 after treated with the compounds [23] , and collected to 15 mL centrifuge tubes. The control group without treatment with compound was also harvested. The tubes were putted into ice for 10 minutes, then spin for 2 minutes at 1,150× g to precipitate the worms. The worm pellets were rinsed three times with cold M9 buffer, air dry, and weighed. The worm pellets were resuspended by using 1 mL methyl ethanol (HPLC grade) (for resveratrol) or H2O (for FUDR) and sonicated 50 times (200 V, operation 5 seconds every 5 seconds). Then, the worm solution was centrifuged under 12, 000× g for 3 minutes. The supernatant of the worm solution containing resveratrol or FUDR was transferred to a 1.5 mL centrifuge tube.
To test the drug metabolism, the worms treated with 400 µM, 200 µM, 100 µM, 50 µM, 25 µM, and 12.5 µM of resveratrol and FUDR, respectively, for 6 hours under NGM dead method were transferred to NGM plates without resveratrol and FUDR. Then, the worms were harvested at the 10 min, 30 min, 1 hr, 2 hr, 3 hr, 4 hr, 6 hr, 8 hr, 12 hr, and 16 hr time points. Subsequent sample preparation was the same as described above.
Free full text: Click here
