Indigo

After successfully engrafting PBMCs in NCG mice, we modeled T-cell tumors by intraperitoneally injecting 2.5 × 10⁶ luciferase-expressing MT2 cells into each NCG-HuPBL mouse (generation of stable luciferase-expressing MT2 ATL tumor cell line is explained in Supplementary Information). NCG-HuPBL mice injected with luciferase-expressing MT2 cells underwent in vivo bioluminescence imaging at various times as specified for each experiment. Luciferase-based bioluminescence imaging was performed with a NightOWL II LB 983 (Berthold) in vivo imaging system. Mice were first anesthetized by inhaling isoflurane and were maintained with 2% isoflurane during imaging procedures. Mice were injected (I.P.) with D-luciferin (prepared in DPBS) at 150 mg/kg and imaged immediately after anesthesia. Images were captured, and bioluminescence intensity and tumor area were quantitated using Indigo live imaging analysis (Berthold). Total flux and area were measured through the automated analysis method by Indigo (Berthold).

Five week old NCG (NOD/ShiLtJGpt-Prkdc^em26Cd52Il2rg^em26Cd22/Gpt) mice were obtained from GemPharmatech (Nanjing, China), and at 6 weeks, they were injected intraperitoneally (i.p.) with 1 × 10⁵ LEL6 cells. 7 days later, the mice were injected with 1 million UTD cells/3B CAR-T cells/3BD CAR-T cells. The second-round and third-round injections of 2 million UTD cells/3B CAR-T cells/3BD CAR-T cells were performed at Day 14 and Day 21 post LEL6 cell injection.
Mice were subjected to weekly bioluminescence imaging. Briefly, mice were anesthetized and injected intraperitoneally (i.p.) with luciferin substrate (150 mg/kg) (Perkin Elmer, Waltham, MA, United States). Luciferase activity was measured within 10 min using NightOW LB983 (Berthold). Data were analyzed and exported using IndiGO (Berthold, Stuttgart, Germany). Luminescence signal intensity is represented by radiance in photons per second per centimeter squared per steradian (p/sec/cm²/sr). Mice were monitored weekly for weight loss and mortality for 70 days. Normally, we assumed that mice die by default when their weight drops by 25%.

NightOWL camera (Berthold Technologies) was used for imaging fluorescence of bacteria grown on agar plates or plant roots. Each CCD image consisted of an array 1,024 by 1,024 pixels, and after acquisition, images were postprocessed for cosmic suppression and background correction. Fluorescence CCD images were acquired with a Ring-light epi illumination accessory exposed for 1 s and special filters have to be used. Filter used for GFP quantification at excitation wavelength 475/20 and emission wavelength 520/10 nm. Filter used for mCherry quantification at excitation wavelength 550/10 and emission wavelength 620/10 nm. Images were analyzed with the imaging software IndiGO (Berthold Technologies).
The author responsible for distribution of materials integral to the findings presented in this article is: Philip S. Poole (philip.poole@plants.ox.ac.uk).