Vivoglo caspase 3 7 substrate

Rats underwent repeat 2D BLI and 3D DLIT imaging at 6 and 24 hours post-ablation following an intraperitoneal injection of VivoGlo^™ Caspase-3/7 Substrate (100 mg/kg; Promega). Z-DEVD–Aminoluciferin is a prosubstrate containing the DEVD tetrapeptide sequence recognized by caspase-3 and -7. The DEVD peptide is cleaved in the presence of activated caspase-3 or -7 thereby liberating the aminoluciferin to react with luciferase and generate light. Thus, the light output is a sensitive and specific measure of real-time intratumoral caspase-3/7 activity (28 (link),29 (link)).

All animal work was conducted in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at The Rockefeller University. All animals were housed and fed using the university’s standard husbandry protocols. For experimental metastasis assay, 4T1, EO771-LM3, MDA-MB-231-LM2, and PDXO cells labeled with a luciferase and GFP reporter or a luciferase and ZsGreen reporter were injected into 6–10 week-old female Balb/c (The Jackson Laboratory, 000651), C57Bl/6 (The Jackson Laboratory, 000664), and immunocompromised NOD-scid-gamma (NSG) (The Jackson Laboratory, 005557) mice respectively via tail vein. Tumor growth was monitored weekly by luminescence imaging using an IVIS Lumina II (Caliper Life Science). Lung nodules were detected by H&E staining. To monitor Caspase 3/7 activity in vivo, the lung bioluminescence signal was measured using VivoGlo Caspase 3/7 Substrate (Promega) and normalized to the bioluminescence signal from D-luciferin (GoldBio). For mammary fatpad growth assays, 150,000 4T1 cells were resuspended in 1:1 mixture of PBS with Matrigel (Corning) and injected into one of the fourth mammary fatpads of mice in a total volume of 100 μL. Tumor size was measure with digital calipers and tumor volume was calculated using the formula widtĥ2 x length x π/6.