Ami imager

The next day after CRISPR electroporation, 1 × 10⁶ NK cells were washed with PBS and injected into NSG mice i.v.; the cells were then supported with 1 μg rhIL-15 i.p. 3 times per week. For K562 tumor challenge experiments, approximately 1.5 × 10⁶ luciferase-expressing K562 cells were injected i.v. 4 days after NK cell injection, and 100 ng/mouse rhIL-15 was used to support NK cells for the duration of the experiment after tumor injection. Bioluminescent imaging (BLI) was performed twice a week on an AMI imager (Spectral Instruments Imaging) 10 minutes after i.p. injection of 150 mg/kg d-luciferin. Quantification of BLI signals was performed using Aura software (Spectral Instruments Imaging).
For proliferation assessment, NK cells were washed with PBS and incubated with 1:2,000 CellTrace Violet (Invitrogen) following the manufacturer’s protocol for labeling before injection into the mice. Dye dilution was tracked at time of mouse harvesting by flow cytometry.
For experiments assessing persistence, proliferation, and ex vivo functionality, NK cells were maintained with 1 μg/mouse rhIL-15 i.p. 3 times per week for the entire course of the study.

FACS sorted and expanded Luciferase⁺GFP⁺ hBM-MSCs were injected into the wound beds or non-wounded skin of anesthetized db/db mice or NSG mice (2.5 × 10⁵ cells/mouse) on the day of wounding unless otherwise indicated. At different timepoints post hMSC injection, luciferase activities were detected in vivo. Mice were injected intraperitoneally with d-Luciferin (0.15 g luciferin per 1 g bodyweight) and imaged 10 min after with IVIS-100 or IVIS Spectrum imaging system (Perkin Elmer, Waltham, MA), or with Ami Imager (Spectral Instruments Imaging, Tucson, AZ, USA) based on instrument availability in different animal housing facilities. Images were obtained and analyzed using manufacturers’ software (Perkin Elmer: LivingImage; Spectral Instruments Imaging: Aura). Bioluminescence was quantified using the LivingImage software (Perkin Elmer).

BxPC-3-luciferase-expressing human pancreatic cell line, which stably express luciferase (JCRB cell bank Cat. No. JCRB1448) were inoculated into the hind flanks of Hsd:Athymic Nude-Foxn1^nu mice (Envigo RMS Inc). Tumors were allowed to establish for 6 days, then treated with cetuximab-IR700 by retro-orbital injection (RO). Localized laser illumination at 50, 150, or 300 J/cm² was applied 24 ± 1 h later. Bioluminescence imaging was conducted on an AMI imager (Spectral Instruments Imaging) at time 0 (pre-light), and 1 or 3 days after the application of light.