Tim 3 mab

Freshly isolated or in vitro-cultured cells were stained with anti-CD3, CD4, CD8, CD14, CD19, CD25, HLA-DR, PD-1, and Tim-3 mAb (all from Biolegend). Staining of CD32 was performed by using two different anti-human CD32 mAb: FUN.2 clone (mouse anti-human CD32 PE or PECy7 conjugated, Biolegend) and IV.3 clone (mouse anti-human CD32 purified antibody, Stem Cell Technologies) that was revealed with a secondary PE goat Fab2 anti-mouse IgG (DAKO). Intracellular detection of Ki-67 antigen with anti-Ki-67 antibody was performed using fixed and permeabilized cells following the manufacturer's instructions (BD Biosciences). Control samples were incubated with an isotype-matched antibody. For the determination of CD32 expression, PE and/or PE-Cy7 mouse IgG2b kappa (Biolegend) were included as isotype controls, using a threshold value ≤ 0.2 in all cases. When CD32 was determined by using unconjugated IV.3 clone, a purified mouse IgG2b kappa (Biolegend) was used as isotype control followed by a secondary PE anti-mouse antibody. Dead cells were excluded by forward and side scatter characteristics. Statistical analyses were based on at least 100,000 events gated on the population of interest. The data were acquired using a FACSCanto II (Becton Dickinson) and analyzed with FlowJo software.