D luciferin

The ELISPOT used to analyze antigen-specific IFNγ secretion of TCR-engineered T cells was performed as described previously [7 (link)]. For measuring cytotoxicity, 1×10⁴ DCs cotransfected with luciferase-encoding IVT RNA and titrated amounts of CXorf61 encoding RNA were co-cultured with TCR-transfected T cells for 3 h. D-Luciferin (BD Biosciences) was added to the cells. One hour later luminescence was measured using a Tecan Infinite M200 reader (Tecan). Cell killing was calculated by measuring the reduction of total luciferase activity. Viable cells were measured by the luciferase-mediated oxidation of luciferin. Specific killing was calculated by following equation:
$\mathit{\text{Specific\hspace{0.17em}killing\hspace{0.17em}in\hspace{0.17em}}}\%=100-\left[\frac{(\mathit{\text{sample}}-\mathit{\text{complete\hspace{0.17em}lysis}}}{(\mathit{\text{max\hspace{0.17em}viable\hspace{0.17em}cells}}-\mathit{\text{complete\hspace{0.17em}lysis}}}\times 100\right]$

In all imaging procedures mice were anesthetized using isoflurane.
For BLI D-Luciferin (Firefly Luciferin, BD) was injected intraperitoneally (150 mg/kg) and mice were imaged using IVES CCD imaging system. The bioluminescent signal intensity in the region of interest was quantified as total light emission using Living Image Software (Caliper Lifesciences).
For PET (Focus 120) mice were injected with radiotracers FDG ([18F]-Fluorodesoxyglucose: 3.72 ± 0.44 MBq; 100 μL in saline) and FET ([18F]-Fluoroethyltyrosin: 3.92 ± 0.12 MBq; 100 μL in saline). Scans were performed 45 min after injection. Analysis of the PET images was performed with the AMIDE software.
MRI was performed using a 9.4 tesla small bore animal scanner and a dedicated mouse quadrature-resonator (Bruker, Germany). The MRI protocol consisted of a localizer and a T2-weighted spin echo RARE (Rapid Acquisition with Relaxation Enhancement) sequence. The RARE sequence in axial orientation featured a FOV of 30 mm²; a matrix size of 256x256 pixels, and an in-plane resolution of 117x117 μm². The slice thickness was 0.5 mm. Tumor volumes were calculated using MIPAV (Bethesda, USA).

Mice were injected intravenously with DOTMA/Chol:RNA (4:1) or DOTMA/DOPE:RNA (1.3:2) RNA-lipoplexes including 20 µg Luc-RNA, and in vivo luminescence imaging was performed as described previously [3 (link)]. Briefly, 6 h after RNA-lipoplex injection, mice were anesthetized with isoflurane (Abbott) in a flexi glass chamber and an aqueous solution of D-Luciferin (BD Biosciences) was administered intraperitoneally. Mice were then placed in the light-tight chamber of IVIS Spectrum In Vivo Imaging System (PerkinElmer), and emitted luciferase signal was acquired for 1 min. Luciferase signal superimposed on a black-white photographic image of the mouse was presented on a rainbow color scale.

HEK293T cells (5 × 10⁵/well) were cultured in 24-well plates one day before transfection, and then co-transfected with 60 ng 3′-UTR luciferase reporter plasmids, 20 ng β-galactosidase plasmids, 20 nM miRNA-223 mimics or the negative controls by using Lipofectamine RNAi MAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. Cells were harvested 24 h post-transfection and further subjected to luciferase and β-galactosidase activitydetection by using the D-Luciferin (BD Biosciences) and o-nitrophenyl-β-d-galactoside (ONPG), respectively. The firefly luciferase and β-galactosidase activities were measured using the Multi-Mode Microplate Reader (BioTek Synergy NEO). The results were normalized as the ratio of firefly luciferase activities to β-galactosidase activities.