agent: Diisononyl Phthalate concentration: 25 mM time: 24 hours gender: Male strain: Sprague Dawley
Treatment protocol
On day 3 of culture, cells were dosed with chemical: Fresh 100X chemical dosing solutions in 1% DMSO were prepared 24 h before exposure for all chemical doses. Cells were treated with two concentrations of chemical or vehicle on the morning of day 3 and harvested for RNA at 24 and 48 h post treatment. Prior to RNA isolation all treatments were examined under a light microscope for signs of visible cytotoxicity (i.e. cell death). Five independent cell-cultures (1 well of 6 well plate X 5) per each time point/dose combination were performed to obtain 5 separate biological replicates.
Growth protocol
Frozen male Sprague Dawley primary rat hepatocytes were purchased from XenoTech (Lenexa, KS) and stored in the vapor phase of liquid N2, until use. Cells were thawed using supplier’s protocol and XenoTech cell isolation kit (Lenexa, KS). Briefly, one, 4.5 ml cryovial containing 17 x 106 rat hepatocytes was removed from liquid N2 and immediately placed in a shaking 37oC water bath for 2 min. Hepatocytes were transferred to a 50 ml conical tube containing 40 ml of solution A (XenoTech Dulbecco’s Modified Eagles Media supplemented with isotonic Percoll, no pen/strep). The cryovial was rinsed with 4.5 ml of Solution B (XenoTech Dulbecco’s Modified Eagles Media supplemented with isotonic Percoll, no pen/strep) and transferred to the 50 ml conical tube containing cells in solution A. Hepatocytes were centrifuged at 60 g (1000 rpm) using an International Equipment Company Centra-8R Centrifuge (Chattanooga, TN) for 5 min at room temperature. Following centrifugation the supernatant was aspirated and hepatocytes were resuspended in 35 ml of Solution B and centrifuged at 60 g (1000 rpm) for another 3 min at room temperature. The resulting cell pellet was resuspended in 30 ml of XenoTech resuspension media (Lenexa, KS) and cell viability was evaluated by trypan blue exclusion. Following the assessment of viability, cells were resuspended in Modified Chee’s culture media (XenoTech, Lenexa, KS) at a concentration of 0.7 - 1.0 x 106cells/ml, and 2 ml of cell suspension was transferred to each well of 6 well collagen coated plates (Corning Corning, NY) and placed in a 37oC incubator (5% CO2, 80% humidity). Following 3 to 4 hours of incubation to allow hepatocytes to attach, media was removed and 2 ml of ice cold modified Chee’s culture media (XenoTech, Lenexa, KS) supplemented with 0.25 mg/ml BD Matrigel, (BD Biosciences, Bedford, MA) was added to each well using pipets chilled to 4oC. Hepatocytes were incubated at 37oC, 5% CO2, media was replaced every day for the next 2 days with Modified Chee’s culture media (XenoTech, Lenexa, KS).
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated at 24 and 48 h post treatment, using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH), further purified using a Qiagen RNeasy kit (Valencia, CA) and RNA integrity was validated using a NanoDrop 8000 Spectrophotometer (Wilmington, DE).
Label
biotin
Label protocol
Labeled cRNA was synthesized from 1 ug of total RNA from the 24 h and 48 h time point samples using the MessageAmpII Biotin-Enhanced labeling kit (Ambion, Austin, TX) and Agencourt RNAClean beads according to manufacturer’s instructions (Beckman Coulter Inc., Danvers, MA).
Hybridization protocol
Twenty micrograms of labeled cRNA was fragmented and hybridized to Affymetrix Rat Genome 230 2.0 array (Santa Clara, CA) for 16 h at 45°C. Hybridized arrays were washed using a GeneChip Fluidics Station 450.
Scan protocol
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000.
Description
Time Course/Dose Response Study
Data processing
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 1500.
