Journal: Archives of biochemistry and biophysics
Article Title: The invertebrate Caenorhabditis elegans biosynthesizes ascorbate
Figure Lengend Snippet: Paraquat and ethanol stress do not increase ascorbate levels in C. elegans (A) A mixed population of animals was obtained from approximately six NGM plates and incubated in S-medium supplemented with OP50 E. coli and 0, 50, 100, and 200 µM paraquat for 7 d as described in the “Materials and Methods.” GC-MS analysis was performed as described in the “Materials and Methods” and the amount of ascorbate in samples was analyzed by comparing the EIC area of ascorbate (332 m/z) to that of serine (204 m/z). The points represent four independent samples, the horizontal line is the average, and the error bars denote standard deviations. Statistical significance was determined using an unpaired, two-tailed Student’s t-test. (B) Mixed populations of animals from 80 NGM plates were incubated in a microcentrifuge tube at 20 °C and 160 rpm in M9 medium supplemented with 0, 1%, 5%, or 10% ethanol. After incubation for 39 h, the animals were pelleted, washed, lysed, and analyzed by reversed-phase HPLC according to the “Materials and Methods” section. The data points represent two independent samples and the horizontal line denotes the average. Statistical significance was determined using an unpaired, two-tailed Student’s t-test.
Article Snippet: Gas chromatography-mass spectrometry (GC-MS) analysis After obtaining the animals as described above, pellets of worms were transferred to a microcentrifuge tubes and 150–300 µl of GC-MS extraction buffer (0.5 mM butylated hydroxytoluene (BHT, Sigma, catalog # B1378) in methanol) was added.
Techniques: Incubation, Gas Chromatography-Mass Spectrometry, Two Tailed Test, High Performance Liquid Chromatography