Cytotoxicity assays

ALL cell killing by NK cells was analyzed using calcein-AM assay purchased from Life Technologies [17]. Target tumor cells (10×10⁶) were labeled with (0.5μM) calcein-AM for 30 minutes at 37°C. Following staining, cells were washed with PBS, counted using Trypan blue (Sigma), and 2×10⁶/mL cells were incubated with VAY736 (20 μg/mL) for 2 hours. After washing 2 times with PBS, ALL (10×10³) cells per well (96 well plate) were co-cultured at 37°C with MACs sorted indicated NK cells (5×10⁴) at 5:1 ratio for 4 hours. NK cell purity was >95 % as determined by CD56⁺, CD3⁻ cells via flow cytometry. The percentage of specific lysis from triplicate wells was determined using the following equation, in which “AFU mean spontaneous release” is calcein-AM release by target cells in the absence of antibody or NK cells and “AFU mean maximal release” is calcein-AM release by target cells upon lysis by detergent. % specific lysis = 100 × (AFU mean experimental release−AFU mean spontaneous release)/(AFU means maximal release−AFU means spontaneous release).