Characterization of Mouse Bone Marrow Cells

Cell surface antigens of mouse BM mononuclear cells (BMMNC) obtained from mice treated or not treated with 8 ppm NO were analyzed by flow cytometry (Becton-Dickinson FACScan, Franklin Lakes, NJ, USA) as described [29 (link)]. Fluorescein isothiocyanate or phycoerythrin (PE)-labeled antibodies were purchased from BD Biosciences (San Jose, CA, USA). Semi-solid cultures were performed using murine BM cells prepared from NO-treated mice, sGC transgenic mice, and human BM cells as described [30 (link)]. Following injections of phenylhydrazine, erythroblast-rich cells were prepared from the spleen [27 (link)] and isolated using magnetic-activated cell sorting columns (MidiMACS separator and Anti-Ter119 microbeads, Miltenyi Biotec Inc., Auburn, CA, USA) [28 (link)]. Mouse globin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and vasodilator-stimulated phosphoprotein (VASP) expression in spleen-derived erythroblasts were determined by immunoblotting as described [31 (link)]. Antibodies used were: anti-mouse β-globin and anti-GAPDH (sc-31116 and sc-25778, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-phosphoVASP (Ser239) (#3114, Cell Signaling Technology, Danvers, MA, USA).