cell line: ES-D3 (ATCC) cell type: murine pluripotent embryonic stem cells (ESC) time: 24 h exposured to: monobenzyl phthalate concentration: 0.00441 mM
Treatment protocol
ESC were exposed to phthalate monoesters (MEHP, MBuP, MBzP, or MMP) in 0.3% DMSO during the differentiation protocol from day 3 onwards (time 0 h). Phthalates were dissolved in DMSO and tested in a concentration-response fashion (0.00441, 0.0143, 0.0441, 0.143, 0.441, 1.43 and 4.41 mM). Concurrent solvent control cultures (0.3% DMSO) were exposed from day 3 onward, and collected after 24 h (day 4, time 24 h). All groups contained 7–8 replicates, individually cultured. EB were collected in RNA protect in a 15 ml tube (Qiagen #76526) and stored at −20 ◦C prior to RNA extraction.
Growth protocol
ES-D3 (ATCC) pluripotent embryonic stem cells (ESC) were cultured in complete medium (CM), consisting of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% foetal bovine serum, 1% non-essential amino acids, 2 mM l-glutamine, 1% 5000 IU/ml penicillin/5000 ug/ml streptomycin , and 0.1 mM b-mercaptoethanol . ESC pluripotency was maintained by adding leukaemia inhibitory factor (LIF), 1000 units/ml to the culture medium. ESC were kept for a maximum of 25 passages and are passaged every other day. Differentiation was initiated by culturing the cells via the ‘hanging drop method’ in which approximately 750 cells/drop were cultured in 56 droplets of 20 ul of CM, on the inner side of a lid of a Petri dish (94 mm × 16 mm), containing 5 ml phosphate buffered saline (PBS), Ca2+ and Mg2+ free . ESC were cultured in a humidified atmosphere (37 ◦C, 5% CO2) for three days during which the cells proliferated and formed cell aggregates (i.e. embryoid bodies (EB)). EB were transferred to a bacterial Petri dish (60 mm × 15 mm) containing 5 ml CM and cultured in a humidified atmosphere (37 ◦C, 5% CO2) for two days. Subsequently each individual EB was transferred to a single well of a 24-wells plate , containing 1 ml CM. Within 5 days aggregates further differentiated and displayed spontaneously contracting cell foci. At day 10, all wells were microscopically examined and scored based on the presence or absence of contracting cells.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted by using the RNA Mini-extraction kit (Qiagen).The concentration of the RNA was measured by Nanodrop (Thermo Scientific). The quality of the RNA was checked using the 2100 Bio-Analyzer (Agilent) and Shimadzu MultiNA. Samples with a RNA Integrity Number (RIN) ≥ 8 were used for microarray analysis.
Label
biotin
Label protocol
Samples were randomized, blinded and further processed for array analysis at ServiceXS (Leiden, The Netherlands) using Affymetrix HT MG 430 PM array plates. Array analysis methods were according to manufacturer’s protocols. For each individual sample 100 ng RNA was used for the labelling reaction. The Affymetrix 3 IVT-Express labelling kit (#901229) was used to synthesize biotin-labelled cRNA. Subsequently, 7.5 ug cRNA of each sample was fragmented using a hybridization cocktail.
Hybridization protocol
Next, 0.0375 ng/ul of cRNA was hybridized on the Affymetrix HT MG-430 PM array. After hybridization, washing and staining was performed with the Affymetrix HWS Kit (#901530).
Scan protocol
The Array plates were scanned via the Affymetrix GeneTitan scanner. Data quality was checked by Affymetrix Expression Console software.
Description
MBzP1.6
Data processing
Data was normalized using the Robust Multichip Average (RMA) algorithm, using an MBNI custom CDF version (http://brainarray.mbni.med.umich.edu) version 13.
