Cytotox 96 cytotoxicity assay kit

Respective sensitivity of BC cells to NK cells was determined using the CytoTox 96 cytotoxicity assay kit (Promega, 017317) according to the manufacturer's instructions. In brief, 1 × 10⁴ BCap37 or MDA-MB-231 target cells were seeded in triplicate at NK cell-to-target cell (E:T) ratios of 10:1, 5:1 and 2.5:1. An anti-NKG2D Ab (50 mg/ml, Novus, Littleton, USA, clone 149810) or control mAb (50 mg/ml, Novus) was added to experiments 1 h before co-culture. After 4 h of incubation, the supernatant was removed for analysis. The following formulas were used to calculate spontaneous and specific cytotoxicity: % specific release=(experimental release−effector spontaneous release−target spontaneous release)/(target maximum release−target spontaneous release) × 100%.

Singularized cells were seeded in a 96-well plate (2,500 cells per well) and either infected with iodixanol-purified wild type H-1PV (MOIs of 1, 10, or 50 PFU per cell) or mock-treated with an equal volume of 40% iodixanol (VisipaqueTM, GE Healthcare, Chalfont St Giles, UK) in Ringer solution. The metabolic activity of the cells was quantified using the WST-1 assay according to the manufacturer’s instructions (Roche Diagnostics, Mannheim, Germany). Absorbance of the dye was measured photometrically at 450 nm, with a reference wavelength of 620 nm.
Cell lysis was determined by measuring the release of lactate dehydrogenase into culture medium by use of the Cytotox 96 cytotoxicity assay kit™ (Promega GmbH, Mannheim, Germany). At the time after infection indicated cells underwent triton lysis applying following the manufacturer’s instructions with pelleting the cells at 400 × g in v-shaped 96-well plates and transfer of the supernatant necessary for the analysis of neurosphere cultures.