Anti cd56 apc cy7

PBMCs from 42 MS patients (22 RRMS, 8 SPMS and 12 PPMS) and 17 controls matched for HCMV serostatus were incubated overnight at 37°C with recombinant IL-2 (200 U/ml). The response of NK cells to the HLA class I-defective 721.221 B-lymphoblastoid cell line with or without rituximab (50 ng/ml) was assessed following a 4-h incubation (E/T = 1/1). A complementary approach was performed using EBV(+) AKBM cells as targets following induction of the lytic cycle in the presence of EBV(+) or EBV(–) sera, as previously described (32 (link), 33 (link)). Surface expression of CD107 as a marker of degranulation and intracellular TNFα production was analyzed by flow cytometry as previously reported (34 (link)), using the anti-CD107-APC (BD Pharmigen) monoclonal antibody during incubation together with monensin (GolgiStop® BD) and brefeldin (GolgiPlug® BD). Cultures were then stained with anti-CD3-PerCP (BD Pharmigen), anti-CD56-APC-Cy7 (Biolegend), and anti-NKG2C-PE (R&D System), permeabilized, fixed and stained intracellularly with anti-TNFα-CFBlue (labeled by Immunostep), anti-FcRγ-FITC (Millipore), and anti-PLZF-PE CF594 (BD Biosciences). Data acquisition was performed with an LSRFortessa cytometer (BD Biosciences).

Bone marrow samples were divided into 2 tubes. The first tube was used to measure the MM burden in bone marrow. Cells were stained with anti-CD45-V500 (662912; BD), anti-CD19-PE-Cy7 (560728; BD), anti-CD138-APC (347193; BD), anti-CD38-V450 (562444; BD) and anti-CD56-APC-Cy7 (362512; BioLegend) for 30 min. After fixation and permeabilization, the cells were then stained with anti-clambda-PE (555797; BD) and anti-ckappa-FITC (555791; BD). Data were collected using FACS Canto II flow cytometer (BD, USA), and a minimum of 1 × 10⁶ cells were acquired per sample. MM cells were analyzed according to the report by the European Myeloma Network [44 (link)]. Minimal residual disease (MRD) was defined according to international consensus guidelines and MRD negativity was defined as less than 0.01% nucleated cells [45 (link)].
The second tube was used to detect BCMA and CD38 expression on MM cells. Cells were stained with anti-CD45-V500 (662912; BD), anti-CD19-PE-Cy7 (560728; BD), anti-CD138-V450 (562935; BD), anti-CD56-PerCP-Cy5.5 (560842; BD), anti-CD38-APC (555462; BD) and anti-BCMA-PE (130-117-544; Miltenyi Biotec). BCMA and CD38 expression was reported as the percentage of positive cells in CD45⁺ CD138⁺ CD19⁻ cells. Data were collected using FACSCanto II flow cytometer (BD, USA), and data were analyzed using FlowJo software (TreeStar, USA).