We followed hematological parameters by CBC through the entire therapy. For extra specifics on clodronate administration schemes please check with Supplementary Fig. 23. Iron supplementation was performed as described in supplementary Fig. 15, 17 and 23. Hematological research Hematological values have been established as previously described57. In brief, we collected blood samples by retro orbital puncture under anesthesia and CBCs had been measured on an Advia 120 Hematology Strategy. Flow cytometry analysis of mouse erythroid cells We harvested BM and spleen cells as previously described57. For erythroid analysis, we incubated single cell suspensions with Fluorescein isothiocyanate labeled anti mouse CD71, Phycoerythrin conjugated anti mouse CD44 and Allophycocyanin conjugated anti mouse Ter119 antibodies for 15 minutes on ice. Samples had been washed with PBS supplemented with 1% BSA and acquired within a FACSCalibur instrument outfitted using a dual laser.
For determination of DNA content, we very first stained the cells together with the cell surface markers, washed after which re suspended in 300ul of diluted DRAQ5 10 15 minutes prior to working. For apoptosis analysis we stained single cell suspensions with PE labeled anti mouse CD71, APC conjugated anti mouse CD44 and Pacific Blue selleck inhibitor conjugated anti mouse Ter119 antibodies for 15 minutes on ice. Right after washing, cells had been incubated with 7AAD and FITC labeled Annexin V in 100ul of 1x binding buffer according to your manufacturers instruction. For cell cycle analysis, we utilized the APC BrdU flow kit, according on the makers guidelines. In short, one mg of BrdU was administered to mice by IP injection and BM and spleen have been harvested 1 hour post BrdU administration.
Single cell suspensions were first incubated with FITC labeled anti mouse CD71, PE labeled anti mouse CD44 and Pacific Blue conjugated anti mouse Ter119 antibodies as described above. Following cell surface stain, we stained cells with 7AAD and APC conjugated anti BrdU antibody as described in selleck chemicals the kit guide. Samples had been run inside a FACS Canto II strategy equipped with 3 lasers. Examination was performed making use of flow jo program. Macrophage and myeloid analysis by movement cytometry BM and spleen cells have been harvested as previously described57. Single cell suspensions were incubated with PE conjugated F4 80 anti mouse antibody and FITC labeled anti mouse CD11b or Gr1 antibody in 30% mouse serum in PBS for 30 minutes on ice. For Vcam1 evaluation, cells were 1st stained with purified anti mouse Vcam1 antibody in PBS, 1% BSA for 30 minutes on ice. Soon after washing, cells have been stained with FITC Goat Anti Rat Ig for 30 minutes on ice then washed once with PBS, 1% BSA. F4 80 staining was performed with PE conjugated F4 80 anti mouse antibody as described above. Samples were washed with 1% BSA in PBS and acquired within a FACSCalibur instrument equipped using a dual laser.