Victor x4 fluorometer

The specific cytotoxicity of the NK-92 cell lines against target cells was determined using the Europium (EuTDA) cytotoxicity assay (DELFIA, PerkinElmer #C135-100) according to the manufacturer’s protocol and as previously described (20 (link)). Briefly, target cells were loaded with an acetoxymethyl ester of the fluorescence-enhancing ligand (BATDA; Perkin Elmer #C136-100) and then co-cultured at 10,000 cells/well in triplicate with effector cells at the indicated E:T ratios. After 2 hours of co-culture, supernatants were collected for measurement of the fluorescent signal reflecting target cell lysis using a VICTOR X4 fluorometer (PerkinElmer). Specific lysis was calculated according to the standard formula in the manufacturer’s instructions.

Intracellular ROS production was evaluated using a DCFH-DA probe (Immunological Sciences, Rome, Italy). The DCFH-DA probe is cell permeable and is subjected to deacetylation by esterase to produce nonfluorescent DCFH, which is retained in the cytosol. In the presence of ROS, DCFH is oxidized to fluorochrome 2′,7′-dichlorofluorescein. Thus, this probe has been utilized as an indicator of oxidative stress in biological systems. Briefly, after treatments, the aged HDFs were incubated with DCFH-DA solution (25 µM) at 37 °C for 30 min. ROS levels were then analyzed using VICTORX4™ fluorometer (PerkinElmer, Waltham, MA, USA) with excitation and emission filters of 488 and 535 nm, respectively. The values obtained were normalized for cell number and expressed as relative fluorescence unit (RFU)/10⁵ cells.