Pharmingen apc

At 90 days following systemic intrasseous administration of mixed UCB donor cells or HUDC, the collected blood samples were washed and suspended in a phosphate-buffered saline (PBS) staining buffer containing 1% bovine serum albumin (BSA). To block nonspecific binding, the blood samples were treated with mouse BD Fc Block^TM Reagent (BD Biosciences) for 5 min, subsequently treated with anti-human monoclonal antibodies (mAbs) at saturating concentrations and incubated for 30 min on ice. The following mAbs were used: CD4 (BD Pharmingen^TM APC-Cy^TM7, RRID:AB_398521, BD Biosciences), CD19 (APC/Cyanine7, RRID:AB_314248, BioLegend, San Diego, CA, USA), CD20 (BD Pharmingen^TM APC Mouse Anti-Human CD20, RRID:AB_398670, BD Biosciences), CD45 (Brilliant Violet 570^TM, RRID:AB_10899568, BioLegend), and HLA-ABC class I (BD Pharmingen^TM APC, RRID:AB_398603, BD Biosciences) to measure the respective marker expression level. Following mAbs incubation, all samples were washed twice, resuspended in a staining buffer, and analyzed by FC (Gallios, Beckman Coulter, Brea, CA, USA) employing a BD LSRFortessa^TM Cell Analyzer (RRID:SCR_018655, BD Biosciences) and Flowjo^TM software (For Mac, Version vX.0.7., RRID:SCR_008520, Becton Dickinson) to assess the in vivo phenotype of peripheral blood samples following HUDC administration.

To assess the biodistribution and presence of human cell-based therapy within the selected organs of NOD SCID mice, FC analysis was conducted in the selected lymphoid organs (bone marrow, lymph nodes, spleen, and thymus), and non-lymphoid organs (brain, intestine, kidney, liver, lung, and peripheral blood) at 90 days following systemic intraosseous delivery. To fulfill this aim, we selected the anti-human mAb HLA-ABC class I (BD Pharmingen^TM APC, RRID:AB_398603, BD Biosciences), which specifically targets the HLA-ABC class I antigen on human cells, to assess the human origin of the cells within the selected organs. The cell solution isolated from the respective organs was stained in PBS containing 1% BSA, with anti-human mAb, and incubated for 40 min at 4 °C in the dark. The samples were washed with PBS supplemented with 1% BSA, fixed with 4% of paraformaldehyde, washed with PBS, and then assessed by a flow cytometer (Gallios, Backman Coulter, CA, USA) using the Flowjo^TM (For Mac, Version vX.0.7., RRID:SCR_008520, Becton Dickinson) software.