Luciferin

Athymic nude mice and NOD/SCID gamma (NOD.CB17-Prkd^scid IL2rg^tm1/Bcgen) mice at about six weeks of age (Envigo) were used to evaluate the treatment for HCC orthotopic tumor growth using MET-CAR-T cells. Briefly, MHCC97H^luc+ cells (5 × 10⁵) were used to initiate subcutaneous (SQ) tumor growth for liver implantation into the host mice to initiate intrahepatic tumor growth. 4–5 days after surgery, mice bearing tumors were randomized into experimental groups (n = 8) according to the bioluminescence imaging (BLI) intensity to receive a one-time treatment of MET-CAR-T cells (5 × 10⁶) through intravenous (i.v.) injection. Intrahepatic tumor growth was measured by BLI once a week. To perform BLI, each mouse received intraperitoneal injection of 100 µl luciferin (15 mg/ml, Biosynth). After 10–15 min, mice under anesthesia with isoflurane were imaged using an optiMAX imager (Precision Medicine). To evaluate the effectiveness of treatment, the average BLI signal intensity at each time point was analyzed with the Student’s t test (p < 0.05). All studies involving animals were performed at ETSU animal facility and are approved by the ETSU Institutional Animal Care and Use Committees.

Tissues were isolated and kept in half-frozen Hank’s Balanced Salt Solution (HBSS). 300 μm brain slices were prepared with a vibratome (Leica VT1200S, Buffalo Grove, IL, USA). Tissues were immediately transferred to tissue culture inserts (EMD Millipore, Billerica, MA, USA) and cultured in 35 mm dishes containing 1 ml of explant medium formulated for equilibration with air (high glucose DMEM [Mediatech, Manassas, VA, USA], 4 mM sodium carbonate, 10 mM HEPES, 52 U/ml penicillin, 52 μg/ml streptomycin, 4 mM L-glutamine, 2% B-27 [GIBCO, Grand Island, NY, USA], 0.1 mM luciferin [BioSynth, Itasca, IL, USA]). When indicated, tissues were treated with 10 μM forskolin for 2 hours in order to enhance rhythmicity.

HEK293T and A549 cells transfected with HT-DNA, ISD45, or vehicle for 6 hours were cocultured with L929-ISRE-LUC cells for 24 hours at 37°C unless otherwise stated. Cells were lysed in LUC lysis buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% NP-40), and cell debris was pelleted by centrifugation at 21,000g for 10 seconds. Supernatant was transferred to a white-walled 96-well plate (Life Science Products) and combined with LUC buffer (20 mM Tricine from MilliporeSigma, 2.67 mM MgSO from Thermo Fisher Scientific, 4.7 H₂O, 0.1 mM EDTA from Thermo Fisher Scientific, 33.3 mM DTT from Thermo Fisher Scientific, 530 μM ATP from Thermo Fisher Scientific, 270 μM acetyl CoA lithium salt from MilliporeSigma, 470 μM luciferin from Biosynth, 5 mM NaOH from Thermo Fisher Scientific, 265 μM magnesium carbonate hydroxide from MilliporeSigma). LUC activity was monitored by a BioTek synergy HTX plate reader.

For the leukemia transplantation model sublethally irradiated C;129S4-Rag2^{tm1.1Flv Il2rgtm1.1Flv}/J knockout mice (JAX stock #014593) [24 (link)] were injected with 1 × 10⁵ MOLM-13 cells expressing control shRNA or CSF2RB shRNA and luciferase into their tail veins. BLI measurements were performed using the IVIS200 imaging system (Xenogen) after intraperitoneal injection of luciferin (150 mg/kg; BioSynth, Staat, Switzerland). Data were quantified with Living Image Software (Xenogen). For subcutaneous tumor model C;129S4-Rag2^{tm1.1Flv Il2rgtm1.1Flv}/J knockout mice were subcutaneously injected with 5 × 10⁶ MOLM-13 cells expressing control shRNA or CSF2RB shRNA into their right flanks. Tumor size was measured by a caliper and calculated as described here [25 (link)]. Tumor weight was measured after sacrificing the mice on day 17. Staining was performed using antibodies against phospho-STAT5 (see above) or CSF2RB (Abcam, Cambridge, UK).

Ishikawa cells were transfected with the reporter gene firefly luciferase (Ishikawa/luc) and treated in vitro either with control siRNA (NC) or PLP2 siRNA (siPLP2). 3 × 10⁶ Ishikawa/luc + NC, Ishikawa/luc + siPLP2 were transplanted intravenously into 4 weeks old female nude mice. After 6 weeks, endpoint measurements were performed using NightOwl LB 981 systems (Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany). Mice were anesthetized and intravenously injected with 150 μL luciferin (27 mg/mL, 200 mg/kg) (Biosynth, Staad, Switzerland). Measurements started 20 min after luciferin injection. For all images an exposition time of 5 min was chosen. After completion of in vivo imaging animals were sacrificed. The intensity of the bioluminescence signal was color coded and overlayed with bright field picture.

Luciferin was added (0.5 mm, Biosynth AG, Switzerland) to 3 ml of medium containing cells in 35-mm glass coverslip culture dishes (Iwaki), and incubated on the microscope stage at 37 °C, 5% CO₂, and 20 or 1% O₂. Imaging was carried out using a Zeiss Axiovert 100 microscope with a Fluor 10 × 0.5 NA objective. The photons emitted by individual cells were collected using a Hamamatsu ORCAII BT 512 CCD camera (C4742-98 Hamamatsu Photonics Ltd, UK) controlled with Metamorph software. A series of images were acquired using a 30-min integration time over 80 h. AQM advanced 6 software (Kinetic Imaging, UK) was used for image analysis with background correction. All these experiments were performed at least three times and in each experiment at least 30 cells were recorded and analyzed.