Detection of NTR Activity in A549 Lung Cancer Cells A549 cells were seeded in T75 culture flasks and incubated in 1% or 20% oxygen atmosphere for 48 hours. A solution of BL660-NTR in DMSO (2 mM, 50 µL) was quickly added into each flask such that the final concentration of the probe is 10 µM and 0.5% DMSO. The cells were incubated with the probe for 60 minutes (under the respective culture conditions), washed with fresh PBS, and detached from the culture flasks. The cells were then transferred to a 15 mL centrifuge tube and pelleted at 1000 rpm for 5 min at room temp. The cells (4.25 × 10 6 cells/mL) were then resuspended in PBS along with 10% protease inhibitor solution (1 protease inhibitor mini tablet per 10 mL PBS, Pierce, Thermo Fisher Scientific) and sonicated on ice for 2.5 minutes (pulse 01, 01, 40%). The cell debris was removed via centrifugation at 4 °C. The cell lysates were further diluted 2.5-fold by PBS and 475 µL were transferred into 24-well plates (n = 4 for each condition), treated with luciferase (25 µL, 0.25 mg/mL), and imaged immediately using the IVIS imaging system. Bioluminescence light was collected in open mode (no filters were applied). ROIs were drawn around each well. The signal intensity was quantified using the Living Image Analysis Software. BL Imaging of Hypoxia in a Lung Cancer Model NU/J mice bearing A549-Luc2 tumors were anesthetized using isoflurane (1-3% for maintenance; up to 5% for induction) in oxygen from a precision vaporizer. After testing to ensure animals are fully under anesthesia, an initial background scan is recorded. BL660-NTR or compound 4 formulated in 3:7 v/v DMSO:PBS (see SI for details) was then administered via retroorbital injection. Images were captured using the IVIS imaging system at 5, 15, 30, 45, 60, and 75 minutes after injection. Bioluminescence light was collected in open mode (no filters were applied). ROIs were drawn around each tumor and summed for each animal. The signal intensity was quantified using the Living Image Analysis Software.