Anti cd34 clone ram34

BMNC staining was performed in medium containing 2 % fetal bovine serum (FBS). All monoclonal antibodies (mAbs) were added at saturating concentrations and the cells were incubated for 30 min on ice, washed twice, resuspended in staining solution at a concentration of 5 × 10⁶ cells/ml, and subjected to analysis using an LSR II (Becton Dickinson, Mountainview, CA). The following anti-mouse Abs were used to detect fluorescein isothiocyanate (FITC)-anti-CD117 (c-Kit) (clone 2B8; BioLegend, San Diego, CA) and Phycoerythrin (PE)-Cy5 anti-mouse Ly-6A/E (Sca-1) (clone D7; eBioscience™, San Diego, CA). All anti-mouse lineage markers (Lin) were conjugated by PE and purchased from BD Biosciences: anti-CD45R/B220 (clone RA3-6B2); anti-Gr-1 (clone RB6-8C5); anti-T-cell receptor β (TCRβ; clone H57-597); anti-TCRγδ (clone GL3); anti-CD11b (clone M1/70); anti-Ter-119 (clone TER-119); and anti-CD34 (clone RAM34) as described [8 –10 (link)].

Single-cell suspensions of whole bone marrow were incubated on ice with conjugated antibodies in PBS containing 2% FCS and 5 mM EDTA (Merck). Dead cells were excluded with Fixable Aqua Dead Cell Stain (Thermo Fisher Scientific). For detection of the progenitor populations granulocyte-macrophage progenitors (GMP), common myeloid progenitors (CMP) and megakaryocyte erythrocyte progenitors (MEP), cells were stained with fluorochrome-conjugated anti-lineage (CD3, clone 17A2; Ly-6G, clone M1/70; CD11b, clone RB6-8C5; CD45R(B220), clone RA3-6B2; Ter-119, clone Ter-119, cat. #133311, Biolegend), anti-CD117 (clone 2B8, Biolegend), anti-CD127 (clone A7R34, Biolegend), anti-Ly6A/E (clone D7, Biolegend), anti-CD16/32 (clone 93, Biolegend) and anti-CD34 (clone RAM34, BD). Flow cytometry analysis was performed on the cytoflex platform (Beckman Coulter) and analyzed with FLowJo v.10 software (TreeStar).