Vitamin c sodium l ascorbate

Eggs were isolated by standard bleach treatment and hatched overnight at room temperature. The resulting L1 larvae were then distributed over NGM plates supplemented or not with 10 mM vitamin C (Sodium L-ascorbate) (Sigma-Aldrich, ST-Louis, MO) and grown at 25°C. A concentration of 10 mM ascorbate was chosen in this study as it has previously been shown that such a concentration did not influence the survival of C. elegans[25 (link)]. The developmental stage of the animals was monitored visually until the worms reached the larvae L4 stage. Animals were precisely staged by observing vulval and gonad formation by Nomarski optics before collecting the animals for analysis as described previously
[39 (link)]. Worms were spun down in an eppendorf tube and lysed in TRIZOL (Invitrogen, Carlsbad, CA) to extract total RNA. RNA quality was checked with an Agilent Bioanalyzer 2100 and libraries were made using the Illumina’s TruSeq v2 kit according to the manufacturer’s recommendations. The RIN (RNA Integrity Number) of all samples was above 8.0. Fifty base pairs paired end sequencing was performed on the HiSeq 2000 machine form Illumina.

NRK49F cells and NRK52E cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (HyClone, Logan, UT) supplemented with 10% fetal bovine serum (FBS) (Gibco, Foster City, CA) and 1% penicillin/streptomycin (HyClone), and the cells were cultured at 37°C in a 5% CO₂ environment. DBP exposure solutions were prepared by dissolving DBP in DMSO and adding this solution to the medium up to concentrations of 1, 10, or 100 μmol/L. The concentration of DBP in solvent (DMSO) did not exceed 0.5% (v/v). Vitamin C [(+)-sodium L-ascorbate] (Sigma Chemical Co., St. Louis, MO, USA) was dissolved in sterile deionized water. The antioxidant dose of vitamin C was determined to be 50 μM. Accordingly, this concentration of vitamin C was added along with DBP to the culture medium. The solutions of DBP and DBP + vitamin C were sterilized by passage through a 0.22-μm filter (Millipore Ireland, Cork, Ireland) before use.