HEK293 cells, SKOV3 cells, OVCAR-3, and ES-2 cells were all maintained in regular DMEM supplemented with 10% (v/v) FBS, penicillin-streptomycin. The antibodies applied in this study include anti-HA antibody (Beyotime, AF0039), anti-Flag antibody (Sigma F1804), anti-CtBP1 antibody (BD biosciences 612042), anti-CtBP2 antibody (BD biosciences, 612044), anti-γH2AX (Sigma-Aldrich, SAB5600038) and normal mouse IgG (Santa Cruz, sc2025). The chemicals include MTOB (4-methylthio-2-oxobutanoate, Sigma-Aldrich, k6000), 2-DG (2-Deoxy-D-glucose, Sigma-Aldrich, D8375), 3-BP (3-bromopyruvate, Sigma-Aldrich, 16490), FK866 (Sigma-Aldrich, F8557), cisplatin (Sigma-Aldrich, PHR1624), metformin (Sigma-Aldrich, PHR1084), penicillin-streptomycin (ThermoFisher), Puromycin (ThermoFisher). Luciferin (Yeasen), Doxycycline (Beyotime).
Luciferin
Manufactured by Yeasen
Sourced in China
Luciferin is a bioluminescent compound that emits light when combined with the enzyme luciferase. It is a key component in various laboratory applications that require light-producing reactions, such as cell-based assays and reporter gene studies.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Doxycycline, by Beyotime (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Fk866, by Merck Group (1 mentions)
Anti γh2ax, by Merck Group (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Anti ha antibody, by Beyotime (1 mentions)
Anti ctbp1 antibody, by BD (1 mentions)
Normal mouse igg, by Santa Cruz Biotechnology (1 mentions)
Metformin, by Merck Group (1 mentions)
T7 high yield rna transcription kit, by Novoprotein (1 mentions)
Hydrogenated soybean phosphatidylcholine hspc, by Lipoid (1 mentions)
1 1 dioctadecyl 3 3 3 3 tetramethylindotricarbocyaine iodide dir, by Yeasen (1 mentions)
Ivis spectrum instrument, by PerkinElmer (1 mentions)
Luciferin, by PerkinElmer (1 mentions)
Ivis imaging system, by PerkinElmer (1 mentions)
5 protocols using luciferin
1
Cell Line Maintenance and Antibody Use
2
Liposome Formulation and mRNA Synthesis
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Hydrogenated soybean phosphatidylcholine (HSPC) was purchased from Lipoid (Newark, USA). Cholesterol was from Nippon Fine Chemical (Osaka, Japan). Deuterated cholesterol was from Cambridge Isotope Laboratories (Tewksbury, USA). 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) was from A.V.T. (Shanghai, China). (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraeb-19-Y13-(4-methylpiperazin-1-Y1) propanoate (H3) was synthesized by BirdoTech (Shanghai, China) (6 ). EZ Cap™ firefly luciferase mRNA was provided by ApexBio (Houston, USA). 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyaine iodide (DiR) and luciferin were obtained from Yeasen (Shanghai, China). Water used through experiments was purified using a Millipore Milli-Q purification system (Bedford, MA). All other reagents were of chemical purity or analytical grade from Sangon Biotech (Shanghai, China).
For the synthesis of OVAL mRNA used in the in-situ hybridization (ISH) study, we used the T7 high yield RNA transcription kit from Novoprotein (Shanghai, China) and performed in vitro transcription (IVT) of a linearized DNA template from plasmid pCl-neo-sOVA (Addgene, Watertown, USA). The RNA was capped and attached poly(A) tails using enzymes included in the kit (Novoprotein, Shanghai, China).
For the synthesis of OVAL mRNA used in the in-situ hybridization (ISH) study, we used the T7 high yield RNA transcription kit from Novoprotein (Shanghai, China) and performed in vitro transcription (IVT) of a linearized DNA template from plasmid pCl-neo-sOVA (Addgene, Watertown, USA). The RNA was capped and attached poly(A) tails using enzymes included in the kit (Novoprotein, Shanghai, China).
3
In Vivo Bioluminescent Imaging of FLuc mRNA-LNP
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FLuc mRNA‐encapsulated 4N4T‐LNPs were prepared and examined. Male BALB/c mice were intramuscularly injected with 20 µg of 4N4T‐FLuc mRNA. After 8 h, the mice were intraperitoneally injected with 3 mg of luciferin (YEASEN) dissolved in 200 µL of PBS. Then, the mice were anesthetized using isoflurane (RWD) and imaged in an In Vivo Imaging System (IVIS) Spectrum instrument (PerkinElmer) 15 min after injection of substrate. To plot the curve of luciferase activity over time, the mice were injected with the substrate and imaged at different time points. The animal experiments in this study have been approved by the Institutional Animal Care and Use Committee (IACUC) of West China Hospital, Sichuan University.
4
Evaluating Anti-Tumor CAR-T Cells in Mice
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Mice were maintained under pathogen-free conditions, and food and water were provided ad libitum. All mice used were females aged 5–6 weeks. To evaluate the antitumor activity of PD-1KD BCMA-targeted CAR-T cells in vivo, female mice were inoculated with 5×106 PD-L1/BCMA+K562 cells intravenously. Approximately 7 days later, 1×107 CAR-T cells or a PBS control were infused intravenously, and animal survival was monitored over time. The mice were anesthetized and injected with 200 µL of luciferin i.p. (15 mg/mL, YEASEN, 40 901ES). Bioluminescence images were acquired 10 min after luciferin injection and analyzed using the IVIS Imaging System and software (PerkinElmer).
5
Visualizing Protein-Protein Interactions
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The CDS of GW6a, CLG1, GW2, and HDR3 were individually cloned into pCAMBIA1300-nLUC or pCAMBIA1300-cLUC to express GW6a-nLUC, CLG1-cLUC, GW2-cLUC, and HDR3-cLUC. The CDS of CLG1, GW2, and HDR3 were cloned into pSuper1300-GFP, respectively, to express CLG1-GFP, GW2-GFP, and HDR3-GFP in tobacco leaves. Each construct was transformed into the Agrobacterium EHA105 cells and co-infiltrated into tobacco leaves as indicated. Tobacco leaves were submerged in a solution with luciferin (YEASEN) for 48 h to capture LUC signals using a CCD imaging system (Tanon).
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