We followed hematological parameters by CBC through the entire therapy. For a lot more 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 were determined as previously described57. In quick, we collected blood samples by retro orbital puncture underneath anesthesia and CBCs were measured on an Advia 120 Hematology Process. Flow cytometry evaluation 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 equipped using a dual laser.
For determination of DNA content material, we initially stained the cells with all the cell surface markers, washed and then re suspended in 300ul of diluted DRAQ5 ten 15 minutes in advance of running. For apoptosis examination we stained single cell suspensions with PE labeled anti mouse CD71, APC conjugated anti mouse CD44 and Pacific Blue selleck conjugated anti mouse Ter119 antibodies for 15 minutes on ice. Following washing, cells had been incubated with 7AAD and FITC labeled Annexin V in 100ul of 1x binding buffer in accordance to your suppliers instruction. For cell cycle analysis, we utilized the APC BrdU movement kit, according to your manufacturers guidelines. In quick, 1 mg of BrdU was administered to mice by IP injection and BM and spleen had been harvested 1 hour submit BrdU administration.
Single cell suspensions have been initially incubated with FITC labeled anti mouse CD71, PE labeled anti mouse CD44 and Pacific Blue conjugated anti mouse Ter119 antibodies as described over. Following cell surface stain, we stained cells with 7AAD and APC conjugated anti BrdU antibody as described in selleck chemical the kit guide. Samples had been run inside a FACS Canto II technique equipped with three lasers. Analysis was carried out working with movement jo application. Macrophage and myeloid analysis by movement cytometry BM and spleen cells were harvested as previously described57. Single cell suspensions have been 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 thirty minutes on ice. For Vcam1 evaluation, cells have been to start with stained with purified anti mouse Vcam1 antibody in PBS, 1% BSA for thirty minutes on ice. Just after washing, cells were stained with FITC Goat Anti Rat Ig for thirty minutes on ice then washed when with PBS, 1% BSA. F4 80 staining was performed with PE conjugated F4 80 anti mouse antibody as described over. Samples were washed with 1% BSA in PBS and acquired inside a FACSCalibur instrument outfitted using a dual laser.