Anti cd56 apc

The ADCC assay for measuring intracellular NK cell IFN-γ and CD107a expression was conducted and analysed with the gating strategy as previously described^{23 (link)}. Briefly, 96-well plates were coated overnight at 4 °C with A/California/04/09 HA protein (1 μg/ml) and chimeric cH9/1 HA protein (1 μg/ml). The plates were then washed with PBS and incubated with heat-inactivated sera (prediluted 1:10) for 2 h at 37 °C. Plates were then washed again with PBS and incubated with 10⁵ CD16 176 v NK-92 cells per well (mycoplasma-free, human NK cell line expressing high affinity 176 V variant CD16 receptor) (Fox Chase Cancer Center, Philadelphia, PA, USA). As a negative control, NK-92 cells lacking the expression of CD16 were added to an additional well per sample. Cells were incubated with anti-CD107a-AF488 antibody (Biolegend, San Diego, CA, USA), Brefeldin A (5 μg/ml, BD) and monensin (5 μg/ml, BD) for 16 h at 37 °C. After incubation, the cells were stained with LIVE/DEAD Fixable Aqua dead cell staining kit (Invitrogen), anti-CD3-PE CF594 (BD) and anti-CD56-APC (BD) before intracellular staining with anti-IFN-γ-BV-421 (Biolegend). The cells were acquired on BD Fortessa. Data analysis was performed using FlowJo version 10 (treeStar).

To investigate the capability of DLE-newly derived NK-like cells of producing IFN-gamma (IFNγ) upon stimulation, an IFNγ assay was performed. After 30-day coculture, the produced cells were stimulated with IL-12 (10 ng/mL) and IL-18 (50 ng/mL) for 12 hours. NK-like cells were harvested and Brefeldin A (BFA, 10 μg/mL) was added for 4 hours to inhibit protein intracellular transport. After BFA blockage, cells were carefully washed, followed by incubation with anti-CD56-APC, anti-CD11c-FITC (BD Biosciences), and anti-IFNγ-PE (Biolegend) conjugated antibodies to perform extra- and intracellular staining, respectively. A FACSCanto II equipment was used for multiparametric flow cytometric analysis.

Samples of EDTA anticoagulated peripheral blood (5 mL) were collected from three groups’ patients. All samples were tested within 6 h of being obtained. Briefly, CD3⁺/CD4⁺/CD8⁺ T-cell, CD19⁺ B-cell, CD3⁻CD16⁺CD56⁺ NK-cell, CD3⁺CD16⁺CD56⁺ NKT-cell, CD4⁺CD25⁺CD127⁻ Treg-cell, CD3⁺HLA-DR⁺ T-cell and CD4⁺HLA-DR⁺ T-cell, CD8⁺ CD28⁺, and CD16⁺CD56⁺CD69⁺ counts (%) were measured by multiple-color flow cytometry with human monoclonal anti-CD3-FITC, anti-CD4-PE, anti-CD8-APC, anti-CD19-PE, anti-CD16-FITC, anti-CD56-APC, anti-CD25-PE, anti-CD127-BV421, anti-HLA-DR-Alexa700, anti-CD28-BV510 and anti-CD69-PE antibodies (BD Multitest) according to the manufacturer’s instructions. The cells were analyzed by flow cytometry system (Becton Dickinson, USA) according to the operating procedure.

Flow cytometry (FCM) was used to detect clusters of immunocytes in patients with MDS. Fresh whole blood samples collected into EDTA (100 μL) were immunostained with anti-CD3-PerCP, anti-CD8-FITC, anti-CD4-PE, anti-CD56-APC, anti-CD16-FITC, anti-CD19-APC, and mouse isotype controls (BD Biosciences, San Jose, CA, USA) in separate Trucount tubes (BD Biosciences, USA) for 30 min at 4°C in the dark, followed by erythrocyte lysis using 2 mL erythrocyte lytic solution (BD PharMingen, San Diego, CA, USA). After centrifugation at 1300 rpm for 5 min, the supernatant was discarded. Cells were washed with phosphate-buffered saline (PBS) and resuspended in 300 μL PBS, and at least 50,000 cells were acquired on a FACSCalibur flow cytometer (BD Biosciences, USA). Analysis was done using CellQuest software version 3.1 (Becton Dickinson, Franklin Lakes, NJ, USA).