Cd150 bv421

The following antibodies were used: mouse lineage cocktail-PE (BioLegend, cat# 78035), mouse lineage cocktail-APC (R&D Systems, cat# FLC001A), c-Kit-FITC (BioLegend, cat# 161603), c-Kit-APC (BioLegend, cat# 135108), c-Kit-PE/Cy7 (BioLegend, cat# 105814), c-Kit-BV421 (BioLegend, cat# 135124), Sca-1-BV605 (BioLegend, cat# 108133), Sca-1-APC (eBioscience, cat# 17-5981-82), Sca-1-BV421 (BioLegend, cat# 108127), Sca-1-PerCP-Cy5.5 (eBioscience, cat# 45-5981-82), CD150-BV421 (BioLegend, cat# 115925), CD150-PE (eBioscience, cat# 12-1501-82), CD150-PE-Cy7 (BioLegend, cat# 115913), CD48-PE/Cy7 (eBioscience, cat# 25-0481-80), CD48-BV711 (BioLegend, cat# 103439), CD48-APC-Cy7 (BioLegend, cat# 103432),CD34-FITC (eBioscience, cat# 11-0341-82), CD135-PE-Cy5 (BioLegend, cat# 135311), CD135-APC (BioLegend, cat# 135310), CD45.1-PerCP-Cy5.5 (BioLegend, cat# 110727), CD45.2-BV421 (BioLegend, cat# 109831), CD45.2-FITC(eBioscience, cat# 11-0454-82), Gr1-PE (BioLegend, cat# 108407), CD11b-APC (BioLegend cat# 101211), CD4-PE (BioLegend,cat# 116005), CD8a-PE (BioLegend, cat# 100707), B220-APC (BioLegend, cat# 103211).

Prior to antibody incubation, cells were stained with viability dye (Zombie™ dye, 1:500, Biolegend) for live cell/dead cell discrimination and incubated with Fc receptor blocking solution (Human TruStain FcX, BioLegend, CA) to prevent unspecific antibody binding. Extracellular antigens were stained using anti-human cluster of differentiation (CD)4-PE-Cy7, CD8-PE, CD14-PE-CF594 and CD19-FITC/PerCP-Cy5.5, CD20-APC-Cy7, CD25-BV605, CD27-PacificBlue, CD38-FITC, CD80-PE-Cy7, CD150-BV-421, major histocompatibility complex class II (MHC-II)-APC (all Biolegend, CA), CD19-PerCp-Cy5.5, CD40-PE-Dazzle, CD69-FITC, CD86-BV421, and CD95-PE (all BD Biosciences, NJ) antibodies. For analysis of intracellular cytokines, cells were permeabilized by adding fixation/permeabilization solution (Cytofix/Cytoperm, BD Biosciences, NJ) and stained with anti-human interleukin (IL)-6-FITC, IL-10-PE/CF594, and tumor necrosis factor (TNF)-Alexa Flour 700 (all BD Biosciences, NJ) antibodies. Apoptosis was evaluated using propidium iodide-PE and annexin V-FITC (both BioLegend, CA). Samples were analyzed using a LSRII Fortessa; FACS Diva (BD Biosciences) and FlowJo software were used to quantify flow cytometric data.