Anti cd20 fitc

The supernatant collected from myoblast cultures secreting anti-CD20 antibody was analyzed in a binding capacity assay. Rituximab (anti-CD20, Hoffmann-La Roche) was used as a binding positive control. Human PBMCs isolated from whole blood using BD Vacutainer CPT Sodium Citrate tubes (BD Biosciences, 362782) were pre-incubated with different concentrations of either supernatant or Rituximab (8–0.25 μg) for 30 min at 4°C. Cells were then washed twice with DPBS and stained for 30 min at 4°C with anti-CD20 FITC (BD Biosciences, 555622) and anti-CD19 PE-CF594 (BD Biosciences, 562321). DRAQ7 staining was used (BD Biosciences, 564904) for the selection of viable cells. Stained cells were analyzed using an LSR Fortessa flow cytometer (BD Biosciences) and data were analyzed using BD FACS Diva (BD Biosciences).

Natural killer cell degranulation activity was tested against the K562 erythroleukemia human cell line. In particular, PBMCs derived from patients and from healthy donors were obtained upon Ficoll separation of heparinized blood samples, and incubated with or without 100 U/mL recombinant human IL-2 (NIH) at 37°C overnight. Cells were then incubated with target cells at an effector:target ratio of 1:3 in a final volume of 200 µl in round-bottomed 96-well plates at 37°C and 5% CO2 for 4 h in culture medium supplemented with anti-CD107a-PE (BD Biosciences Pharmingen, San Diego, CA, USA) monoclonal antibody. Cells were then surface-stained with FITC anti-CD3, PC5 anti-CD56, FITC anti-CD14 (Beckman Coulter), FITC anti-CD20, and APC/Cy7 anti-CD16 (BD Biosciences Pharmingen, San Diego, CA, USA) Ab for 30 min at 4°C. The cells were washed, and the proportion of CD3⁻ CD14⁻ CD20⁻ CD56⁺ cells expressing CD107a was analyzed immediately on LSR Fortessa Flow Cytometer (BD) using FACSDiva v6.1.3 software (BD Biosciences, Mountain View, CA, USA). Final analysis was performed using FloJo v.10.2 (TreeStar). The threshold to define CD107a expression in cells co-cultured with K562 target cells (in the presence or absence of IL-2) was set up on cells cultured with IL-2 alone, without K562 cells.

To detect intracellular production of IFN-γ, PBMCs from patients and healthy donors were incubated overnight at 37°C with IL-12 (0.5 ng/ml), or IL-12 (0.5 ng/ml) and IL-18 (0.1 ng/ml) combined. Surface staining was done by incubating the cells with FITC anti-CD3, PC5 anti-CD56, FITC anti-CD14 (Beckman Coulter), FITC anti-CD20, and APC/Cy7 anti-CD16 (BD) mAbs for 30 min at 4°C. Cells were then washed, fixed, and permeabilized with BD Cytofix/Cytoperm kit (BD Biosciences Pharmingen). IFN-γ production was detected by subsequent intracellular staining with PE-conjugated anti-IFN-γ (BD Biosciences Pharmingen). After washing, the proportion of CD3⁻ CD14⁻ CD20⁻ CD56⁺ cells expressing IFN-γ was immediately analyzed on LSR Fortessa Flow Cytometer (BD) using FACSDiva software (BD). Final analysis was done using FloJo v.10.2 (TreeStar).

Cells were harvested from the model by a 3-min trypsin treatment, resuspended in a cell culture medium, and washed with PBS before Annexin V/propidium iodide (PI) assay (Immunostep, Barcelona, Spain) or staining with PE anti-CD19 (Becton Dickinson, Franklin Lakes, New Jersey, USA), FITC anti-CD45 (BD, 345808), FITC anti-CD20 (BD, 345792), and PE anti-CD105 (Caltag Laboratories, Carlsbad, USA, MHCD10504) conjugated antibodies, according to the manufacturers’ recommendations.
A FACS Canto II (BD) cytometer was used and 10,000 events/sample were analyzed using the FACS DIVA Software v8 0.2.