Anti cd5

Single cell suspensions of splenocytes or peripheral blood were prepared using the standard techniques. Fc receptors were blocked by using 2.4G2 mAb prior to surface staining with the indicated antibodies. The negative selection of NK cells was determined by staining with cell surface monoclonal antibodies (mAbs), including anti-CD19, anti-CD4, anti-CD8a, anti-CD5, anti-Gr1, and anti-Ter-119 (Miltenyi Biotech). The surface phenotype of NK cells was determined by staining with mAbs, including anti-CD3-PE and anti-NK1.1-APC (Biolegend, San Diego, CA). Intracellular IFNγ was measured by pacific blue-conjugated anti-IFNγ (Biolegend) as described previously [47 (link)]. Dead cells were excluded by propidium iodine (PI) staining and live cells were gated on PI-negative cells. The NK cells of donors or recipients were distinguished by using anti-CD45.1 mAb (A20, BD Biosciences, San Jose, CA), FITC-34-2-12 (H-2D^d mAb, BD Biosciences), or Bio-KH95 (anti-H-2D^b mAb, BD Biosciences). Data acquisition was performed with FACS Calibur flow cytometer (BD Pharmingen, San Jose, CA) using a CellQuest software (BD Biosciences). Data analysis was performed using a FlowJo software [48 (link)].

PC5-conjugated anti-CD19 and anti-CD5 were from Miltenyi; PE-conjugated anti-HLA-G1 MEM-G/9 was obtained from Exbio, Praha; and PE-conjugated anti-CD3 was from Beckman Coulter. Biotin-coupled anti-CD4 and PC5-conjugated anti-biotin antibody were from Miltenyi. Purified PC5- and PE-conjugated isotype controls were from Miltenyi. For flow-cytometry analyses, Fc receptors were blocked by a 30-min incubation with 1 μg/μl of pooled purified isotype antibodies in PBS_1x. All staining steps were performed on ice or at less than 4°C and isotype-matched control antibodies were systematically used. Flow-cytometry analyses were performed on a Canto II cytometer (Beckton Dickinson) using FlowJo software (Tree Star).