Polysomal redistribution of mRNAs during restimulation of primed Th2 cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE4500 | GEO | 2006/03/23
SECONDARY ACCESSION(S): PRJNA94499
REPOSITORIES: GEO
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Dishes were incubated with fibronectin solution (16.7 µg/ml) for 30 min in the incubator, washed once with PBS and used thereafter. Isolation of TS cells from post-implantation (E6.5) embryos Embryos were dissected in PBS on day E6.5 and transferred in PBS/10% (v/v) FBS and kept on ice. Before dissociation, embryos were washed in PBS twice. Whole conceptuses were transferred into an U-bottom 96-well plate and incubated in 40 µl trypsin-EDTA (PAN-biotech) for 15 min in the incubator. The content of the well was pipetted up and down several times and transferred to a 24 well plate on pre-plated irradiated MEFS (gammaMEFs) in either TS medium with 1.5x FGF4 and heparin concentration (TS+1.5xF4H) or TX medium with 1.5x FGF4 and heparin (TX+1.5xF4H). TS cell colonies appeared after 2-4 days. Immunofluorescent staining, western blotting. Before staining, cells were washed with PBS once and fixed for 10 min with 4% paraformaldehyde (Sigma-Aldrich). Permeabilization of the cells was performed with 0.5 % (v/v) Triton X-100 (Sigma-Aldrich) in PBS for 10 min and subsequent incubation for 30 min in blocking buffer (2% bovine serum albumin [Sigma-Aldrich], 0.1% (v/v) Triton X-100 in PBS). Samples were incubated overnight at 4°C with the following primary antibodies: Cdx2 (1:200, Cdx2-88, BioGenex Inc), Eomes (1:500,ab23345, Abcam), Tfap2c (1:300, sc-8977, Santa Cruz). After three washing steps, detection was performed with appropriate Alexa Fluor-conjugated antibodies (Invitrogen) at a 1:500 dilution in blocking buffer for 1 h at room temperature in the dark. After secondary antibody incubation, nuclei were stained with Hoechst (Sigma-Aldrich). Cells stained without primary antibodies served as controls. Western blotting was performed with 23 µg of cell lysates from TS cells cultured in TS and TX media by SDS-polycrylamide gel electrophoresis and blotted onto PVDF membranes. Membranes were blocked with 5% (v/v) BSA or 5% (v/v) non-fat milk powder in TBST (TBS with 0.1% Tween). Membranes were incubated with primary antibodies at room temperature for 2 h, washed three times with TBST and labeled with HRP conjugated antibodies (Dako) for 1 h at room temperature. After three washing steps with TBST, membranes were incubated in ECL substrate (Thermo Scientific). RNA isolation and analysis. RNA was isolated from cells using the RNeasy Minikit (Qiagen) according to the manufacturer's protocol. 500 ng total RNA was used for cDNA synthesis by RevertAidPremium (Thermo Scientific). For gene expression microarray analysis, RNA quality was tested using the Agilent 2100 Bioanalyzer (Agilent Technologies). 100 ng total RNA were used for cDNA synthesis. Biotin labeling and fragmentation were performed according to the Affymetrix user manual. cRNA was hybridized for 16h to Mouse Genome 430 2.0 GeneChip expression arrays (Affymetrix). 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