Pe or vio770 labelled anti cd56 mab

This work benefited from umbilical cord blood units (UCBs) and the expertise of Prof. John De Vos, in charge of the Biological Resource Center Collection of the University Hospital of Montpellier—http://www.chu-montpellier.fr/en/platforms (BIOBANQUES Identifier—BB-0033-00031). NK cells were expanded as previously described^{31 ,32 (link)}. Briefly, UCBMCs or PBMCs were isolated through density gradient centrifugation using Histopaque-1077 (Sigma). Blood samples were diluted at 1:1 ratio with RPMI then layered above 10 mL Histopaque in a 50 mL conical tube. Once centrifuged for 30 min at 400×g, the white layers of mononuclear cells (MCs) were collected and washed. Using EasySepTM Human CD3 Positive Isolation kit (StemCell Technologies), the CD3⁺ cell fraction (T and NKT cells) of the MCs was depleted in each sample to better culture the NK cells. Once depletion was verified through flow cytometry, cells were cultured for 20 days. NKs were cultured with γ-irradiated PLH at a 1:4 (NK cell: feeder cell) ratio and IL-2 (100 IU/mL) and IL-15 (5 ng/mL). Feeder cells and cytokines were refreshed every 3–4 days. To monitor expansion, NK cells were stained with APC-labelled anti-CD3 mAb and PE or Vio770-labelled anti-CD56 mAb (both form BD Biosciences). Culture viability was determined at regular intervals through flow cytometry analysis.

This work benefited from umbilical cord blood units (UCBs) and the expertise of Prof. John De Vos, in charge of the Biological Resource Center Collection of the University Hospital of Montpellier—http://www.chu-montpellier.fr/en/platforms (BIOBANQUES Identifier—BB-0033-00031). NK cells were expanded as previously described^{24 ,26 (link)}. Briefly, UCBMCs or PBMCs were isolated through density gradient centrifugation using Histopaque-1077 (Sigma). Blood samples were diluted at 1:1 ratio with RPMI then layered above 10 mL Histopaque in a 50 mL conical tube. Once centrifuged for 30 min at 400×g, the white layers of mononuclear cells (MCs) were collected and washed. Using EasySep Human CD3 Positive Isolation kit (StemCell Technologies), the CD3⁺ cell fraction (T and NKT cells) of the MCs was depleted in each sample to better culture the NK cells. Once depletion was verified through flow cytometry, cells were cultured for 20 days. NKs were cultured with γ-irradiated PLH at a 1:4 (NK cell: feeder cell) ratio and IL-2 (100 IU/mL) and IL-15 (5 ng/mL). Feeder cells and cytokines were refreshed every 3–4 days. To monitor expansion, NK cells were stained with APC-labelled anti-CD3 mAb and PE or Vio770-labelled anti-CD56 mAb (both form BD Biosciences). Culture viability was determined at regular intervals through flow cytometry analysis.