-1b and 1 ng/ml IFN-c. Subsequently, 56105 FL/BM cells were centrifuged with 50% retroviral supernatant at 1100 g for 17855348 90 min in 8 mg/ml polybrene or on Retronectin-coated plates. One day later, lethally irradiated mice were injected intravenously with at least 26105 transduced FL/BM cells. Hematopoietic and lymphoid purchase Aphrodine organs from transplanted mice were analysed at 612 weeks posttransplant or whenever they developed disease. Results from FL and BM transplanted mice were pooled for statistical analysis. Design and Methods This research was reviewed and approved by the St. Vincent’s Animal Ethics Committee. AEC# 012/10. Mice All animal experiments were performed in accordance with the St. Vincent’s Hospital Animal Ethics committee. Either CD45.1 or CD45.2 C57BL/6 mice between 812 weeks old were used throughout this study. Mice were monitored daily and control and Fli-1 mice were sacrificed simultaneously by cervical dislocation when Fli-1 mice became moribund. Foetal Thymic Organ Culture FL reconstitution of FTOCs was performed as described previously. The reconstituted foetal thymic lobes were placed on 0.8 mm polycarbonate membranes floating on 2 ml complete IMDM and analysed by flow cytometry 14 days later. Antibodies Antibodies used for surface staining included anti-TCR , anti-CD4, anti-CD25, anti-CD44, and anti-CD8 . Lineage depletion for CD25/44 staining was accomplished by staining cells with a cocktail of biotinylated mAb and gating out SA-PeCy7. OP9-DL1 Co-cultures E15 foetal liver cells were cultured on OP9-DL1 cells for 6 days in 5 ng/ml FLT3L and 0.25 ng/ml IL-7. FL cells were retrovirally transduced with either MigR1 control or Fli-1. Four days later the GFP+ cells were analysed for presence of DN1 4 progenitors by flow 9504387 cytometry as described above. Flow Cytometry Cell suspensions from mouse BM, thymus, spleen, lymph node or liver were washed in PBS with 2% FBS and 0.01% NaN3. Fc receptors were blocked with 2.4G2. Cells were then stained with primary antibodies for 20 min at 4uC and washed in 200 ml FACS buffer. Staining with streptavidin secondary reagents was performed in an identical manner. Labelled cells were run on a Becton Dickinson FACScalibur or LSR II, and analysed using FlowJo software. Routinely, 36104 to 26105 events were collected. Dead cells were excluded using FSC and SSC gates and with propidium iodide where possible. Intracellular flow cytometry was performed by washing cells in 0.03% saponin in FACS buffer. Cells were then stained with anti-NOTCH1 or isotype control in SAP FACS and after washing, run on a FACScalibur or LSRII analysis. Routinely, 36104 to 26105 events were collected. Histology Fresh tissues were fixed in Bouin’s fixative overnight and then placed into 70% ethanol and embedded in paraffin. Five mm sections were cut and, after standard histological procedure for dehydration, stained with haematoxylin and eosin. Western Blot Analysis Cells were lysed in RIPA buffer with protease inhibitors and equal amounts of total protein per sample were separated via 10% SDS-PAGE and transferred to Immobilon-P transfer membrane for western blotting. Proteins were detected using primary antibodies against FLI-1 and beta-ACTIN and secondary HRP conjugated antibodies followed by visualisation using ECL reagents. Plasmids MigR1-GFP was a gift from Warren Pear and was used as described previously. Fli-1 cDNA was cloned from 129Sv/J embryoid bodies, ligated into MigR1 and the insert confirmed by sequencing. Fli-1 O