Anti cd56 pe

NKCC was assessed using a modified version of the protocol described by Godoy-Ramirez et al. (13 (link)). Briefly, K562 target cells were cultured either alone or with NK cells at an effector:target (E:T) cell ratio of 10:1 for 4 h at 37°C in a humidified 5% CO₂ atmosphere. After incubation, cells were pelleted and resuspended in PBS/1%BSA containing 0.3 µg anti-CD56 PE (Miltenyi Biotec). Following a 10-min incubation on ice, samples were washed in PBS and stained for 5 min with 125 nM of the dead cell stain sytox blue (Life Technologies) prior to flow cytometric analysis.
To measure NKCC, the number of lysed K562 target cells (defined as sytox blue positive) in a total population of 2000 was recorded. From here, the percentage of specific cell lysis was calculated as: (TL-SL/2000) × 100, where TL is the number of lysed target cells in NK-K562 co-culture samples and SL is the number of K562 cells that underwent lysis when cultured with media alone. To measure the target cell–NK cell conjugate formation rate, conjugates were defined as K562 cells exhibiting positive PE fluorescence and the number in a population of 2000 K562 cells was recorded.

MALC obtained at day 10 of growth, were co-cultured with NK cells at ratio E:T 0.5:1 or 1:1 and treated or not with mAbs at 10 μl/ml during 4 h in presence of brefeldin A at 10 μl/mL (except of CD69 detection). Cells were then washed, fixed with 2% PFA during 4 min, washed and permeabilized with 1% saponin during 8 min. Cells were stained with anti-CD56-PE (Miltenyi Biotec), anti-CD3-PE/Cy7 (Beckman Coulter, Roissy, France), anti-CD69-PE-Cy5 (BD Biosciences), anti-perforin (PFN)-FITC (BD Biosciences), anti-IFN-γ-Alexa fluor 647 (BD Biosciences), anti-granzyme B-Alexa647 (BD Biosciences) antibodies during 30 min at 4°C in the dark. Cells were analyzed by flow cytometry with a LSRII flow cytometer (BD Biosciences).

For flow cytometry analysis, cells were washed with PBS containing 10% FBS and incubated for 30 minutes at 4°C for labeling with anti-CD3-FITC (BD Biosciences, clone OKT3), anti-CD56-PE (Miltenyi Biotec, clone MEM-188), anti-CD57-APC (Biolegend, clone NK-1), anti-CD16-BV421 (BD Biosciences, clone 3G8), anti-NKG2C-BV510 (BD Biosciences, clone 134591) and 7AAD. For each sample, 100.000 events were recorded using a FACS Canto II cytometer (BD Bioscience). Cell populations were analyzed using FlowJo v.X.0.7 (TreeStar Inc.). Viability of thawed CB-NK cells was quantified using the LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Invitrogen) following the manufacturer’s instructions.