Anti cd45.2 pe clone 104 monoclonal antibodies

Cell suspensions from embryos at different stages were injected into irradiated adult recipients (CD45.1/1) either directly (suspensions from AGM region or E14.5 foetal livers) or after culture (E11.5 AGM region explants), along with 80,000 CD45.2/1 bone marrow carrier cells. Recipients were irradiated by a split dose (600+550 rad with 3 h interval) of γ irradiation. Donor-derived chimerism was monitored in blood at different time points after transplantation using LSRFortessa (BD). The peripheral blood was collected by bleeding the lateral tail vein into 500 µl of 5 mM EDTA/PBS, and erythrocytes were depleted using PharM Lyse (BD). Cells were stained with anti-CD16/32 (Fc-block), CD45.1-APC (clone A20) and anti-CD45.2-PE (clone 104) monoclonal antibodies (eBioscience). Appropriate isotype controls were used. Dead cells were excluded using 7AAD (eBioscience).

CD45.2/2 cells were injected into irradiated 2- to 3-month-old Bl/6J CD45.1/2 heterozygous recipients along with 100,000 CD45.1/1 nucleated bone marrow carrier cells per recipient. Recipients were γ-irradiated using two doses (600 + 550 rad) separated by 3 h. For day 9.5 culture, one embryo equivalent (e.e.) of the 7 day cultured cells was injected. E11 AGM-sorted cells were injected after 5 days of OP9-coaggregate culture at a dose of 1 e.e./recipient. Donor-derived chimerism was evaluated in peripheral blood at 6 and 14 weeks post transplantation. Erythrocytes were lysed using Pharm Lyse (BD Biosciences), and non-specific binding was blocked with an anti-CD16/32 (Fc-block) followed by staining with anti-CD45.1-APC (clone A20) and anti-CD45.2-PE (clone 104) monoclonal antibodies (eBioscience). Cell populations were identified as CD45.1-PE+ (donor), CD45.1-PE+CD45.2-APC+ (recipient), CD45.1-APC+ (carrier) using FACSCalibur or Fortessa (BD Biosciences).
HSC numbers were assessed using extreme limiting dilution analysis (ELDA) analysis (Hu and Smyth, 2009 (link)). Multilineage donor-derived hematopoietic contribution in recipient blood and organs was determined by staining with anti-CD45.1-V450, anti-CD45.2-V500, and lineage-specific anti-Mac1-fluorescein isothiocyanate (FITC), Gr1-PE CD3e-APC, B220-PE-Cy7 monoclonal antibodies (BD Pharmingen).