Ami ht

For the bioluminescence
assessment, mice were anesthetized with isoflurane gas (2% isoflurane
in oxygen, 1 L/min) during injection and imaging procedures. Intraperitoneal
injections of D-luciferin (Biosynth AG) were done at a dose
of 150 mg/kg, providing a saturating substrate concentration for Fluc
enzyme (luciferin crosses the blood–brain barrier). Mice were
imaged in a light-tight chamber using an in vivo optical imaging system
(AMI HT; Spectral Instruments imaging) equipped with a cooled charge-coupled
device camera. During image recording, mice inhaled isoflurane delivered
via a nose cone, and their body temperature was maintained at 37 °C
in the dark box of the camera system. Bioluminescence images were
acquired between 10 and 20 min after luciferin administration. Mice
usually recovered from anesthesia within 2 min of imaging.

Fluc⁺ P815 cells were counted and plated at a concentration of 10³ cells per 96 wells and mixed with CD3⁻CD7⁺CD56⁺ primary human NK cells at an E:T ratio of 10:1. All NK cells were preincubated with human IL-2 (Life Technologies) at a concentration of 1 µg/ml for 72 h. After 4 h in the BLI killing assay, luciferase expression was detected by adding D-luciferin (Promega). As controls, target cells were left untreated or treated with 2% Triton X-100 in cell-specific media. In some conditions, target cells were treated with anti-CD16 antibody (clone 3G8, mouse IgG1,κ, 10 µg/ml; BioLegend), anti-NKG2D antibody (clone 149810, mouse IgG1, 10 µg/ml; R&D Systems), anti-SIRPα (clone 2H7E2, mouse IgG1, 10 µg/ml; antibodies-online), or anti-CD56 (clone NCAM1/784, mouse IgG1, Abcam, 10 µg/ml). Signals were quantified with Ami HT (Spectral Instruments Imaging) in p/s/cm²/sr.

After the MEF feeder cells attached and were 100% confluent, miPSCs were grown on MEF in KO DMEM 10829 with 15% KO serum replacement, 1% glutamine, 1% MEM with nonessential amino acids, 1% penicillin/streptomycin (pen/strep; all Gibco), 0.2% β-mercaptoethanol, and 100 U leukemia inhibitory factor (both Millipore). Cells were maintained in 10-cm dishes, medium was changed daily, and the cells were passaged every 2–3 d using 0.05% trypsin-EDTA (Gibco). For luciferase expression, 10⁵ miPSCs were plated in one gelatin-coated 6-well plate and incubated overnight at 37°C at 5% CO₂. The next day, medium was changed, and one vial of Fluc lentiviral particles expressing the luciferase II gene under the reengineered EF1a promotor (Gen Target) was added to 1.5 ml medium. After 36 h, 1 ml cell medium was added. After an additional 24 h, a complete medium change was performed. After 2 d, luciferase expression was confirmed by adding D-luciferin (Promega). Signals were quantified with Ami HT (Spectral Instruments Imaging) in maximum photons per second per centimeter square per steradian (p/s/cm²/sr).