For cellular viability assessment, the BM-MSC cells were washed with PBS twice, and then were trypsinized. The harvest cells were stained with CD90-FITC (BD) and CD105-APC (R&D system). In the case of co-culture assays the harvested MNC and CD34+ cells were stained with CD45-FITC (BD) and CD105-APC. In the case of MPN cell lines assays the harvested cells were previously labeled with CD90-FITC and CD45-APC, and then performed the annexin-V kit, as described below.
Cells from the different experimental conditions were stained with Annexin V-PE using the PE annexin V apoptosis detection kit (BD) following manufacturer's instructions. For cell cycle, the cells were stained with propidium iodite (PI), using the kit cycle tests (BD), according to manufacturer's recommendations. Samples were acquired on a FACSCalibur flow cytometer (BD) and analyzed using the Infinicyt software (Cytognos, Salamanca, Spain). Cell-cycle was analyzed using the ModFit LTTM Macintosh program (Verity Software, USA).
Cells from the different experimental conditions were stained with Annexin V-PE using the PE annexin V apoptosis detection kit (BD) following manufacturer's instructions. For cell cycle, the cells were stained with propidium iodite (PI), using the kit cycle tests (BD), according to manufacturer's recommendations. Samples were acquired on a FACSCalibur flow cytometer (BD) and analyzed using the Infinicyt software (Cytognos, Salamanca, Spain). Cell-cycle was analyzed using the ModFit LTTM Macintosh program (Verity Software, USA).