Primary human NK cells elicit limited biosensor activation without IL-2 stimulation. Primary and IL-2–activated human NK cells were assessed for biosensor activation with K562 biosensors at an E:T of 3:1. Human NK cells were negatively selected from healthy donor PBMCs by magnetic bead isolation and confirmed to be >60% CD3^– CD56⁺ by flow cytometry. NK92 was used as a positive control. (A) NK and 24-hour IL-2–stimulated NK are cytotoxic against K562 targets. Error bars represent SD from quadruplicate wells. One representative experiment of 2 is shown. (B) NK and 24-hour IL-2–stimulated NK are unable to activate the GLS.VGPD sensor in K562 clonal lines at an E:T of 0.75:1 (left), although GLS.DEVD activation (right) was observed by 90 minutes after stimulation with IL-2. One representative experiment of 3 with and without IL-2 stimulation is shown. (C) To maximize the biosensor signal, isolated NK were conjugated at a higher E:T (3:1) and the read extended to 240 minutes. NK cells were unable to activate GLS.VGPD (left) and GLS.SGR (middle), although consistent late activation of GLS.DEVD (>90 min) was observed (right). (D) After 6 days’ culture in 1000 U/mL IL-2, NK expanded from PBMCs readily activated GLS.VGPD (left), GLS.SGR (middle), and GLS.DEVD (right) expressed in K562 clonal lines at 3:1. The fold activation for 1 representative experiment of 3 is shown.