D luciferin

NSG mice were purchased from Jackson Laboratory and maintained by the Animal Resource Center at COH. Mice were housed in a pathogen-free animal facility according to institutional guidelines. All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC: 15020). NSG mice (12–16 week old female; 5/group for statistical power) were challenged (IV) with luciferase-expressing tumor cells followed by treatment with CAR T cells(25 (link)). Mock T cell or PBS-treated mice were controls. Tumors were monitored by bioluminescent imagining. Briefly, mice were challenged with 2.5×10⁵ Nalm-6-BAFF-R-KO + 1×10⁵ Nalm-6-CD19-KO or 1×10⁵ Nalm-6-BAFF-R-KO + 1×10⁵ Nalm-6-CD19-KO where indicated. 9–10 days post-tumor challenge, mice were assessed for engraftment and randomly allocated to treatment groups. A single infusion of 2.5×10⁶ CD4 T cells + 1×10⁶ of CD8 T cells of dual-targeting CAR T cells or 1×10⁶ BAFF-R/CD19 dual CAR, CD19 or BAFF-R single CAR-T cells as indicated, were administered intravenously. Mice were anesthetized with isoflurane and received subcutaneous d-luciferin (150 mg/kg; Life Technologies) 10 minutes before imaging with LagoX imaging systems (Spectral Instruments Imaging, Tucson, Arizona, USA). Investigators were not blinded to group allocation or outcome assessment.

For the ATP assay, age-synchronized L1s were transferred to culture plates with food and allowed to develop for 24 h to the late L2/early L3 stage. Larvae were rinsed from culture plates and 50 larvae were dispensed using a Biosort to each well of white 96-well plates (Greiner Lumitrac). Nematodes were exposed to compounds for 24 h with untreated animals reaching the L4 stage, as described for the development assay. After exposure, green fluorescence and luminescence were measured using a FLUostar Optima plate reader (BMG Labtech Inc.). Fluorescence was read at $485\text{nm}$ excitation and $520\text{nm}$ emission. To measure ATP, luminescence buffer [ $0.1\text{mM}$ D-luciferin (Life Technologies), 1% DMSO, 5% Triton-X (Fluka BioChemika), $0.1\text{mM}$ citric acid (Sigma-Aldrich), and $0.2\text{mM}$ dibasic sodium phosphate (Sigma-Aldrich)] was injected into each well. After 3 min, luminescence was read at $570\text{nm}$ using the Optima plate reader.