Luciferin substrate

In vivo experiments were done in accordance with the HMRI institutional guidelines for the use of laboratory animals. To determine tumorigenicity of regenerated SP cells, 8- to 10-week-old NOD/SCID IL2rg−/− mice (Jackson Laboratory, Bar Harbor, ME, USA) (six mice per group) were injected subcutaneously with 0.1 × 10⁶ SP or NSP cell populations isolated from the RPMI8226 GL cell line and suspended in Matrigel (BD Biosciences). Tumor engraftment was monitored weekly by whole-body bioluminescence imaging. Luciferin substrate (Gold Bio Technology, St. Louis, MO, USA) was injected intraperitoneally (IP) and whole-body imaging performed on the Xenogen IVIS system (PerkinElmer, Waltham, MA, USA), as previously reported.^{20 (link)} Mice were euthanized on day 35 in accordance with institutional guidelines. Bioluminescence signal intensity representative of tumor sizes was quantified by Living Image 3.1 (PerkinElmer).
To determine the ability of SP and NSP cells to infiltrate the mouse bone marrow (BM), mice (same as above) were injected via the lateral tail vein with 0.2 × 10⁶ sorted SP or NSP cells. Tumor engraftment was monitored using bioluminescence for 3 weeks. Mice were then euthanized and bone marrow cells were flushed with PBS from femora and tibiae using a 25-gauge needle. The percentage of GFP-positive cells was determined by flow cytometry.

Compound treated NK cells were co-cultured with Ovcar3 cells stably expressing a luciferase plasmid (pLenti CMV puro LUC, Addgene) in white bottom 96-well plates for 2 hours. After incubation, luciferin substrate (GoldBio, 16.6 μg /mL) was added to the plate for 2 minutes at room temperature. Luminescence was then measured using a Spectramax Gemini dual-scanning microplate spectrofluorimeter (Molecular Devices). The results of the co-culture were normalized to TC alone wells to calculate % cell death.