Anti cd56 pe

NPM1^mut-specific CTL products were characterized for phenotype by monoclonal antibody staining and flow cytometry. Anti-CD3 FITC, anti-CD4 PE, anti-HLA-DR PE, anti-CD8 APC, CDγδ FITC, anti-CD56 Pc5.5, anti-CD14 FITC, anti-CD56 PE, anti-CD3 Pc5.5, anti-CD19+ CD20 APC, anti-CCR7 FITC, and anti-CD45RA PE (Becton Dickinson, Franklin Lakes, NJ, USA) were employed.

Analysis of MICA expression on patient-derived PCs was performed by gating the CD38⁺CD138⁺ PC population using the antibodies anti-MICA (clone 159227, R&D Systems, Minneapolis, MN, USA), anti-CD38/APC, and anti-CD138/FITC (both from BD Bioscience, San Jose, CA, USA) as previously reported (16 (link)); samples were acquired using a FACSCanto (BD Biosciences, San Jose, CA, USA) and a FACSCalibur (Becton Dickinson). Analysis of NKG2D and DNAM-1 on NK cells from PBMCs or BM aspirates was performed by gating on the CD45⁺CD138⁻CD3⁻CD56⁺ population using the antibodies anti-CD3/allophycocyanin-H7, anti-CD56/PE, anti-CD138/FITC, anti-CD45/PE-Cy7, anti-NKG2D/APC, or anti-DNAM-1/APC (BD Bioscience). In some experiments, anti-CD16/PerCP mAb was also used (BD Bioscience). In the experiments relative to Figure 4 and Figure S2 in Supplementary Material, all the patients-derived samples were acquired using a FACSCanto (BD Biosciences, San Jose, CA, USA). In the experiments relative to Figure 5, PBMCs derived from healthy donors were incubated with medium alone or serum derived from MGUS or MM patients for 16 h. After harvesting, cells were stained with antibodies from BD Bioscience: anti-CD3/PerCP, anti-CD56/APC, and anti-NKG2D/PE; samples were acquired using a FACSCalibur (Becton Dickinson).
Data analysis was performed using the FlowJo 9.3.2 program (TreeStar, Ashland, OR, USA).

This was undertaken according to our previously published methods (33 (link)). The method utilized whole blood analysis with red cell lysis using Easylyse (BD Biosciences) and with anti-CD56 PE, anti-CD16 FITC, anti-CD3 PE Cy5 and anti-CD69 APC (all supplied by BD Pharmingen). Flow cytometric analysis was undertaken on a Facscalibur (BD Biosciences), and using Cellquest Pro software. A minimum number of 10,000 cells were counted.

Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation through Ficoll-Hypaque of blood samples obtained at the time of allograft biopsy, and stored at—80°C until analyzed. After thawing, 0.5–1 X 10⁶ PBMC were incubated with various antibody combinations for 30 min at 4°C. The fluorochrome-conjugated monoclonal antibodies used were anti-CD19 V500, anti-CD3 V450, anti-CD56 PE, anti-CD14 PE-Cy7, anti-CD45 APC, anti-CD38 PE-Cy7 from BD Biosciences, anti-CD24 APC from Miltenyi Biotec and anti-IgD FITC and anti-CD27 PE from Beckman Coulter. Samples were analyzed with Canto II cytometer (BD Biosciences) and the data were processed using FlowJo software (Tree Star, Ashland, USA).