All the experiments and handling of animals were performed following approval by Kobay A.S. Animal Research and Ethics Committee (Approval number:2019/354). Male CD-1 nude mice (23–24 g, 6–8 weeks old) were maintained in IVCs at 22 ± 3 °C, 55% relative humidity and under a 12 h dark/light cycle. Mice were allowed free access to food and water. Xenograft tumor model was developed by the subcutaneous injection of 1 × 107 A549 cells in 100µL PBS coated with matrigel (1 mg/mL) to the dorsal flank of mice. When the tumor diameter reached about 0.5 cm, mice were separated into 4 groups to apply spherical-shaped nanoparticles (SN) (n = 3), rod-shaped nanoparticles (RN) (n = 3), elliptical disk-shaped nanoparticles (EDN) (n = 3), and polymer solution (n = 3) as control group. Nanoparticles (mPEG-PLGA-FKR560; 18 mg/mL) were injected intravenously through lateral tail vein of mice. Mice were sacrificed and dissected at 16 h post-injection. Fluorescent intensity of tissues was recorded using Newton 7.0 (Vilber, Collégien, France), under 580 nm filter. Mean fluorescence intensity of nanoparticles was calculated using the ImageJ Fiji program (Madison, WI, USA) [38 (link)].
Newton 7
Manufactured by Vilber
Sourced in France
Newton 7.0 is a compact and versatile laboratory instrument designed for precise spectrophotometric analysis. It features a high-resolution display, intuitive interface, and advanced data processing capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Duo lite luciferase assay system, by Vazyme (1 mentions)
Biostack ready, by Agilent Technologies (1 mentions)
Dual luciferase reporter assay kit, by Vazyme (1 mentions)
Cytation 5, by Agilent Technologies (1 mentions)
D luciferin potassium salt, by GoldBio (1 mentions)
In fusion premix, by Takara Bio (1 mentions)
Dual luciferase reporter gene assay kit, by Yeasen (1 mentions)
10 protocols using newton 7
1
Investigating Nanoparticle Biodistribution in Xenograft Mice
2
Transcriptional Activation of MdWRKY87 in Tobacco
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The MdWRKY87 ORF sequence without stop codon was cloned into the pBD-VP16 vector (Han et al., 2016 (link)). The reporter vector contained a GAL4-luciferase (LUC) containing five copies of the GAL4-binding element and a minimal CaMV35S promoter at the 5’ end of the LUC gene (Han et al., 2016 (link)).The effector vectors or reporter vectors were introduced into A. tumefaciens strain GV3101. The A. tumefaciens cells was re-suspended to an OD600 of 1.0 using the buffer (10 mM MgCl2, 10 mM MES-KOH, pH 5.6; 200 μM acetosyringone). A. tumefaciens cells harboring effector vector and reporter vector were mixed 1:1, then injected into the tobacco (N. benthamiana) leaves by using a 1 mL needleless syringe. After spraying 1 mM luciferin onto the leaves, luciferase imaging was performed using NEWTON 7.0 (VILBER LOURMAT, Paris, France). An assessment of LUC and REN activities was conducted using the Duo-Lite Luciferase Assay System (DD1205–01, Vazyme, Nanjing, China) and BioStack Ready (BioTek Instruments Inc., Winooski, Vermont, USA). LUC/REN ratio was used to calculate the results. The primers used for construction are listed in Supplemental Table S1
3
Bioluminescent Infection Imaging in Mice
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Plasmid pBEN276 (54 (link)) was employed to construct a stable reporting system within the chromosome of ExPEC RS218 (25 (link)) for extended trial in vivo. The 5 × 108 CFU bioluminescent RS218 (confirmed by pretest) was intraperitoneally injected into 4-week-old female BALB/c mice (n = 5/group). After 1 h, the animals were intraperitoneally injected with 100 μl of 25 μg gcIFN-20 or PBS. At 4 h post bacterial injection, the mice were anesthetized and transferred to the IVIS (Newton 7.0, Vilber Lourmat, France). The luminescence was tested with an exposure time of 5–15 sec. The imaging system translates the data into false color images that depict the area of strong luminescence with red and mild luminescence with blue. The bioluminescence intensity is proportional to the number of bacteria. The total flux of the target area was quantified by the IVIS software.
4
Bioluminescent Infection Imaging in Mice
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Plasmid pBEN276 (54 (link)) was employed to construct a stable reporting system within the chromosome of ExPEC RS218 (25 (link)) for extended trial in vivo. The 5 × 108 CFU bioluminescent RS218 (confirmed by pretest) was intraperitoneally injected into 4-week-old female BALB/c mice (n = 5/group). After 1 h, the animals were intraperitoneally injected with 100 μl of 25 μg gcIFN-20 or PBS. At 4 h post bacterial injection, the mice were anesthetized and transferred to the IVIS (Newton 7.0, Vilber Lourmat, France). The luminescence was tested with an exposure time of 5–15 sec. The imaging system translates the data into false color images that depict the area of strong luminescence with red and mild luminescence with blue. The bioluminescence intensity is proportional to the number of bacteria. The total flux of the target area was quantified by the IVIS software.
5
Tumor Growth and Metastasis Evaluation in Mouse Xenograft Model
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MDA‐MB‐231‐LUC cells were cultured to 70% confluence, and 2 × 106 cells in 100 μL PBS, were implanted s.c. into the mammary fat pad of 19 female 6‐week‐old NSG (NOD.Cg‐PrkdcscidIl2rgtm1Wjl/SzJ) female mice. Mice were then injected s.c. twice a week (starting from the day of cell injection) with 82.5 mg·kg−1 APPI‐3M‐HSA (10 mice per group) or with PBS (9 mice per group) as a control. Once a week, mice were injected i.p. with 150 mg·kg−1 luciferin, and tumor and metastasis formation were followed using NEWTON 7.0 (Vilber, Collégien, France). At the treatment endpoint, on day 25, all mice were injected i.p. with 150 mg·kg−1 luciferin 10–15 min before they were euthanized. Mice organs were then harvested and scanned for bioluminescence using NEWTON 7.0 to evaluate the metastatic burden (exposure time 30 s, binning 2, f‐stop 0.7). In addition to metastatic potential, the in‐vivo local effect of treatment was assessed qualitatively both macroscopically and microscopically, i.e., in terms of the ease of surgical excision of the primary tumor bulk and histopathological evidence (see next section) of muscle invasion, respectively.
6
Transient Expression Assay in Tobacco
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The ORF of PsTOE3 was obtained and was inserted into binary vector pBI121. Meanwhile, the promoter of PsEBB1 was cloned into pGreenII0800-LUC vector. The recombinant plasmids were introduced into Agrobacterium GV3101 and cultured at 28°C in LB liquid medium with kanamycin and rifampicin. The bacterial cells were injected into N. benthamiana leaves. Four days after injection, the dual luciferase assay was performed for enzyme activity determination in leaves. FLUC and REN (Renilla luciferase) were assayed using a Dual-Luciferase Reporter Assay Kit (Vazyme), and their activities were measured using an ultrasensitive multifunctional microplate reader (Cytation 5, BioTek) and a plant in vitro fluorescence detector (Newton7.0, Vilber). The activity of REN was considered as the reference to normalize the activity of FLUC. Each treatment involved five biological replicates and three technical replicates.
7
Effector/Reporter-based Transactivation Assay
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pGreenII 0800-LUC and pGreen 62-SK vectors were used for the effector/ reporter-based transactivation assay as described previously (Hellens et al., 2005 (link); Fujikawa and Kato, 2007 (link)). The 2.0-kb promoter regions of PtORE1 and PtSGR1 were cloned into the pGreen II 0800-LUC vector (effector). The coding regions of PtRD26 and PtRD26IR were cloned into the pGreen 62-SK vector (reporter). Equal volumes of the suspensions were mixed and infiltrated into N. benthamiana leaves, which were placed in the dark for 24 h and then transferred into the light for 48 h, and detected by a CCD camera (Vilber NEWTON7.0).
8
Dual-Luciferase Transient Expression Assay
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pCAMBIA1300-nLUC and pCAMBIA1300-cLUC vectors were used for SLCA (Chen et al., 2008 (link)). The coding regions of PtRD26 and PtRD26IR were cloned into double digested vectors (KpnI/SalI) using In-Fusion Premix (Lot#1710367A, TAKARA). Overnight-cultured A. tumefaciens GV3101 cells (BC308-01, Biomed, China) harboring the constructed plasmids were resuspended (OD600 = 0.5) in infiltration buffer (10 mM MES pH5.8, 10 mM MgCl2, and 100 μM acetosyringone) for 3 h in dark before infiltration. Equal volumes of the nLUC and cLUC suspensions were mixed and infiltrated into N. benthamiana leaves, which were placed in the dark for 24 h and then transferred into the light for 48 h. Before fluorescence detection by a CCD camera (Vilber NEWTON7.0), the leaves were sprayed with 0.32 mg/mL d -Luciferin potassium salt in 0.1% Triton X-100 (Gold Biotechnology).
9
Transient expression assay for BrpHMA2::LUC in tobacco
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BrpHMA2 fused to the firefly LUC gene (BrpHMA2::LUC) was constructed in pGreenII 0800-LUC as a reporter, and BrpNACs or other AREBs were constructed in the pGreenII 62-SK vector as effectors. pGreenII 62-SK and pGreenII 0800-LUC vectors were used for transient expression assays in tobacco leaves. The reporter and effector were transiently expressed in tobacco leaves mediated by Agrobacterium GV3101 (pSoup). Agrobacterium-infected plants were cultivated under low irradiance for 24 hours and then transferred to light for an additional 2 days [45 (link)]. The LUC signal in the transfected leaves was detected with a CCD camera (Vilber Newton 7.0).
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BrpHMA2 fused to the firefly LUC gene (BrpHMA2::LUC) was constructed in pGreenII 0800-LUC as a reporter, and BrpNACs or other AREBs were constructed in the pGreenII 62-SK vector as effectors. pGreenII 62-SK and pGreenII 0800-LUC vectors were used for transient expression assays in tobacco leaves. The reporter and effector were transiently expressed in tobacco leaves mediated by Agrobacterium GV3101 (pSoup). Agrobacterium-infected plants were cultivated under low irradiance for 24 hours and then transferred to light for an additional 2 days [45 (link)]. The LUC signal in the transfected leaves was detected with a CCD camera (Vilber Newton 7.0).
10
Dual-Reporter System for Transcription Factor Activity
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To detect the transactive activity of MYC2, MYC3, MYC4 and ZAT18, a dual reporter system was constructed. The coding sequences of MYC2, MYC3, MYC4, ZAT18 or their mutants were cloned into the pGreenII 62-SK vector, and the 1-kb promoters of ZAT18 or EDS1 were cloned into the pGreenII 0800-LUC vector (Hellens et al., 2005) . The primers used are given in Table S1. The constructs were transformed into A. tumefaciens strain GV3101(pSoup), which were co-infiltrated into leaves of 4-wk-old Nicotiana benthamiana plants. Leaves were harvested at 36 h post infiltration and were imaged using a charge-coupled device (CCD) camera (Newton 7.0; Vilber, Paris, France). The LUC and REN activity were measured using a Dual Luciferase Reporter Gene Assay Kit (Yeasen Biotech Co. Ltd, Shanghai, China). The LUC : REN ratio represented the relative activity of the promoter.
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