Cd34 clone ram34

For LSK cell analysis, cells were stained for 30 min on ice with antibodies against c-Kit (clone 2B-8), Sca-1 (clone D7), CD16/32 (FcγR II/III; clone 93), Flk2 (CD135; clone A2F10; all from eBioscience), CD34 (clone RAM34; BD Biosciences), and antibodies against Lin markers (Miltenyi Biotech). For erythroid progenitor cells analysis, BM and spleen cells were stained with antibodies against CD71 (clone R17217) and Ter119 (clone Ter119; all from eBioscience). FITC-BrdU Flow Kit was purchased from BD Pharmingen. BM-differentiated Mk’s were stained with PE-conjugated anti-mouse CD41 (clone MWReg30; BioLegend). For quantification of BM Mk’s frequencies, BM cells were stained with anti–CD41-PE and anti–CD45-FITC (Miltenyi Biotech). For intracellular staining of cytokines, BM cells were fixed and permeabilized with BD Citofix/Citoperm fixation and permeabilization solution (BD Biosciences) and stained with CD41-PE, anti–IL-6 FITC (clone MP5-20F3; BioLegend) and anti–TNF-α APC (clone MP6-XT22; BioLegend). 7-aminoactinomycin D solution (BD Pharmingen) was used for cell survival analysis. All the samples were acquired with a Beckman Coulter FacsDiva flow cytometer. Non-stained samples and relative isotype controls were used to set the correct analytical gating. Off-line data analysis was performed using a Beckman Coulter Kaluza version software package.