Fetal livers were resuspended in PBS/ 0.3% BSA and passed through a 40 µm cell strainer (BD Biosciences). Fetal liver cells (FLC´s) were treated with 10 ml Red Blood Cell Lysis Buffer (Sigma-Aldrich) to remove erythrocytes. For sorting TER119− erythroid progenitors, FLC´s were incubated with rat antibodies against the following surface markers: GR1, CD41, CD11b, CD14, CD45R/B220, CD4, CD8 (BD Pharmingen), Ter119 (gift from Albrecht Müller, Julius-Maximilians-University, Würzburg, Germany) and with YBM/42 (gift from Suzanne M. Watt, University of Oxford, Oxford, UK) for 20 min at 4°C. After washing cells were incubated for 20 min at 4°C with anti-rat antibody-coupled magnetic beads and negatively sorted with MACS columns according to the manufacturer's instructions (Miltenyi Biotech). Sorted CFU-E were cultivated for 12-14 h in IMDM (Invitrogen), 30% fetal calf serum (FCS), and 50 µM µ-mercaptoethanol supplemented with 0.5 U/ml Epo (Cilag-Jansen).
Growth protocol
E13.5 Balb/c mouse embryos were dissected from the uterus of sacrificed females
Extracted molecule
total RNA
Extraction protocol
RNeasy Mini Plus Kit (Qiagen, Hilden, Germany)
Label
Biotin
Label protocol
Biotinylated cRNA was generated from 1.5 µg total RNA using the One-Cycle Target Labeling Assay (Affymetrix).
Hybridization protocol
15 µg labelled cRNA were hybridized onto the Mouse Genome 2.0 GeneChip® Arrays (Affymetrix, CA, USA) for 16h at 45°C using Hybridization Wash and Stain Kit (Affymetrix)
Scan protocol
Arrays were washed and stained with GeneChip Fluidics Station 450 and scanned with GeneChip Scanner 3000 using the GeneChip Operating Software (Affymetrix).
