Pe anti cd16 cd56

Duplicate samples of human PBMCs (10⁶/sample) were stained with the following regents for 30 min at room temperature: fluorescein isothiocyanate conjugated anti-CD 4 (FITC-anti-CD4), phycoerythrin conjugated anti-CD8 (PE-anti-CD8), peridinin-chlorophyll-protein conjugated anti-CD3 mAb (PerCP-anti-CD3; Clone SK7/SK1/SK3; BD Tritest; San Jose, CA, USA), PE-anti-CD19, FITC-anti-CD138, PE-anti-IgD, FITC-anti-CD3, and PE-anti-CD16 + CD56+ (BD Bioscience; San Jose, CA, USA). Samples stained with FE-anti-IgG, FE-anti-IgG1, FE-IgG2a, and FITC-anti-IgG (BD Bioscience) were used as isotype controls. After staining, the cells were washed with PBS and analyzed using a FACSAria II flow cytometer (BD BioSciences; Franklin Lakes, NJ, USA). A minimum of 50,000 events per sample were analyzed using FlowJo software (v5.7.2) (FlowJo LLC; Ashland, OR, USA) [13 ].

Analysis of peripheral blood lymphocyte subpopulations by flow cytometry was performed at the diagnosis of CD. Peripheral blood samples were collected and stained using the whole blood lysis technique. Phenotypic analyses were performed by flow cytometry using BD multitest CD3/CD8/CD45/CD4 reagent and CD3/CD16⁺ CD56/CD45/CD19 reagent. These antibodies are labeled as follows: PerCP-anti-CD45, FITC-anti-CD3, APC-anti-CD4, PE-anti-CD8, APC-anti-CD19, PE-anti-CD16 +CD56 (all from BD Biosciences USA). The percentages of the lymphocyte subsets including CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells, CD19⁺ B cells, and CD3⁻CD16⁺CD56⁺ Natural killer (NK) cells, as well as CD4⁺/CD8⁺ ratio were measured.