Adenoviruses using CMV promoter expressing murine LIFR gene or green fluorescent protein (GFP) (Ad-LIFR and Ad-GFP) were constructed by Genechem Company. Overexpression of LIFR or GFP in the liver of HFD mice were achieved by means of tail vein injection of Ad-LIFR or Ad-GFP (1 × 109 plaque-forming units [pfu] for each mouse). Adenovirus particles carrying LIF or GFP (Ad-LIF and Ad-GFP) were multipoint-injected into iWAT on both sides of HFD-induced obese mice (3 × 107 pfu on each side, Genechem Company). Mice were sacrificed at day 14 post injection in the fed state.
Ad gfp
Manufactured by Genechem
Sourced in China
Ad-GFP is a recombinant adenovirus vector that expresses the green fluorescent protein (GFP). It is a tool used for gene expression studies and cell labeling applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ad kdm3a, by Genechem (2 mentions)
Enhance infection solution, by Genechem (2 mentions)
Ad nox4, by Genechem (2 mentions)
Enhanced transfection solution, by Genechem (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Osteogenic differentiation medium, by Merck Group (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Polybrene, by Beyotime (1 mentions)
Ad cd9 gfp, by Genechem (1 mentions)
Adeno x virus purification kit, by BD (1 mentions)
Adshrna, by Genechem (1 mentions)
Pentobarbital sodium, by Merck Group (1 mentions)
Virus purification kit, by Biomiga (1 mentions)
Interferin reagent, by Polyplus Transfection (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
18 protocols using ad gfp
1
Overexpression of LIFR and GFP in Obese Mice
2
Adenovirus-mediated Sik1 overexpression in rats
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Ad-Sik1 and negative control adenovirus containing green fluorescent protein (Ad-GFP) were purchased from Gene Chem Co., Ltd. (Shanghai, China). Ad-Sik1 and Ad-GFP were obtained with a titre of 1×1011 plaque forming units (PFU) /ml. The recombinant adenovirus was stored at –80°C until use. Construction of both vectors was described in S1 Appendix . Sik1-overexpressing rats were established by an injection of Ad-Sik1 or Ad-GFP at an optimized dose of 5×109 PFU in 50μl (diluted with physiological saline) via tail vein once a week for 8 weeks according to the manufacturer’s protocols. Meanwhile, the rats of the control and model groups received physiological saline at the same dosage by tail vein injection.
3
STC2 Modulation in Obesity Mice
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Male C57BL/6 mice aged 8 weeks were purchased from the Shanghai Laboratory Animal Company (Shanghai, China). ob/ob mice were purchased from Nanjing Biomedical Research Institute of Nanjing University (Nanjing, Jiangsu Province, China). HFD-induced obese mice were maintained with free access to HFD (D12492; Research Diet, New Brunswick, NJ, United States) for 12 weeks, and control mice were fed with normal chow diet (NCD) (D12450B; Research Diet). STC2 recombinant protein was purchased from Shanghai Boyi Biotechnology Company (Shanghai, China). For systemic STC2 treatments, ob/ob mice received daily intraperitoneal (i.p.) injections of recombinant STC2 protein (0.5 mg/kg). Adenoviruses expressing murine STC2 gene or green fluorescent protein (GFP) (Ad-STC2 and Ad-GFP) were constructed by Genechem Company (Shanghai, China). Overexpression of STC2 or GFP in the liver of ob/ob mice was achieved by means of tail vein injection of Ad-STC2 or Ad-GFP [2 × 109 plaque-forming units (pfu) for each mouse]. All animal experiments were conducted in accordance with the guidelines of Animal Care Committee of Shanghai Jiao Tong University School of Medicine.
4
Adenoviral Transduction of GFP and Nox1
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Recombinant adenoviral vectors harboring green fluorescent protein (Ad-GFP) or NADPH oxidases 1 (Ad-Nox1) were obtained from Genechem (Shanghai, China). Adenovirus (108 TU/ml) was added to the growth media.
5
Pharmacological Upregulation of P2X7R in Intracerebral Hemorrhage
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S-adenosyl-l -methionine, a CBS-specific agonist (SAM, Sigma-Aldrich, St. Louis, MO, USA), and sodium hydrosulfide, a classical exogenous H2S donor (NaHS, Aladdin, Shanghai, China), were diluted to concentrations of 100 mg/kg [14 (link), 15 (link)] and 5.6 mg/kg [7 (link)], respectively, in vehicle (saline) solution. The rats were treated intraperitoneally with SAM, NaHS or vehicle at 0.5 h after ICH. For the 72-h study, SAM, NaHS and vehicle were administered three times at 0.5, 24, and 48 h after ICH.
For in vivo adenovirus administration, the following two types of recombinant adenoviruses were used for gene transfer: (1) a replication-deficient human adenovirus containing the rat P2X7R (adenovirus P2X7R, Ad-P2X7R), which was used to upregulate P2X7R expression, and (2) an adenovirus containing human GFP (Ad-GFP), which served as a control for Ad-P2X7R. Ad-P2X7R (6 × 1010 pfu/mL) and Ad-GFP (2 × 1010 pfu/mL) were produced by Genechem in Shanghai, China. Both adenoviruses were stored at − 80 °C until use and were diluted to 1.3 × 1010 pfu/mL in an enhanced transfection solution (Genechem, Shanghai, China) before intracerebroventricular injection. A total volume of 10 μL was used for each adenovirus administration to the animals.
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S-adenosyl-
For in vivo adenovirus administration, the following two types of recombinant adenoviruses were used for gene transfer: (1) a replication-deficient human adenovirus containing the rat P2X7R (adenovirus P2X7R, Ad-P2X7R), which was used to upregulate P2X7R expression, and (2) an adenovirus containing human GFP (Ad-GFP), which served as a control for Ad-P2X7R. Ad-P2X7R (6 × 1010 pfu/mL) and Ad-GFP (2 × 1010 pfu/mL) were produced by Genechem in Shanghai, China. Both adenoviruses were stored at − 80 °C until use and were diluted to 1.3 × 1010 pfu/mL in an enhanced transfection solution (Genechem, Shanghai, China) before intracerebroventricular injection. A total volume of 10 μL was used for each adenovirus administration to the animals.
6
Isolation and Culture of Rat Bone Marrow Stem Cells
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Rats were sacrificed by an overdose of chloral hydrate to obtain rat bone marrow stem cells (rBMSCs) for culture. These cultures were prepared according to the protocol developed in Caplan Laboratory and previously carried out in our laboratory (Ke et al., 2016 (link)). The experimental procedures were approved by the Ethics Committee for Animal Research at Zhejiang University (ethics approval number: ZJU20200075). In brief, the tibia and femur, without attached tissues, were excised under sterile conditions. Subsequently, bone marrow was extracted using an injection of basal growth medium (BGM) consisting of α-MEM medium (HyClone, UT, United States) with 10% fetal bovine serum (FBS; Gibco, New York, United States), 100 U/ml penicillin, and 100 μg/ml streptomycin. rBMSCs from passages 3–5 were used in this experiment. When cells were needed for differentiation into osteocytes, the rBMSCs were cultured in an osteogenic differentiation medium supplemented with 10 mM β-glycerophosphate, 50 μg/ml ascorbic acid, and 10–8 M dexamethasone (Sigma-Aldrich Co., St. Louis, United States). Besides, adenoviral vectors encoding a green fluorescent protein (Ad-GFP; GeneChem Co. Ltd., Shanghai, China) were used at a multiplicity of transduction of 100 for a clear presentation of the morphology of BMSCs.
7
Adenovirus-Mediated RRM2 Overexpression
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Cells were seeded in a 6-well plate. Polybrene (4 µg/ml; Beyotime Institute of Biotechnology) was added, followed by RRM2-overexpressing recombinant adenovirus labeled with green fluorescence protein (AdRRM2; cat. no. GOSA0296619; Shanghai GeneChem Co., Ltd.) and negative control adenovirus labeled with green fluorescence protein (AdGFP; cat. no. ADCON267; Shanghai GeneChem Co., Ltd.) according to the manufacturer's standard protocol when cell confluence reached ~40%. After transfection at 37˚C for 8-12 h, the medium was changed to OM. Green fluorescence was observed and images were captured 24 h after treatment using a fluorescence microscope (Olympus IX53; Olympus Corporation; magnification, x100). siRRM2 was purchased from Shanghai GeneBio Co., Ltd. The sequences of siRRM2 were: Forward, 5'-GAGUACCAUGAUAUCUGGCAGAUGU-3' and reverse, 5'-ACAUCUGCCAGAUAUCAUGGUACUC-3'. According to the manufacturer's recommended protocol, siRRM2 was configured as a working system with an ultimate concentration of 20 µM and cells were transfected using Lipo8000 Transfection Reagent (cat. no. C0533; Beyotime Institute of Biotechnology) when cell confluence reached ~40%. In the control group, only Lipo8000 was added without siRRM2. Subsequent experimentation was performed after transfection with recombinant adenovirus or siRNA for at least 2 days.
8
Adenoviral Delivery of KDM3A Upregulation
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Adenoviral vectors encoding KDM3A (Ad-KDM3A) and control Ad-green fluorescent protein (GFP) (Ad-GFP) were constructed by GeneChem Co., Ltd. (Shanghai, China). At 50%-60% cell confluence, equal amounts of Ad-KDM3A or Ad-GFP at 50 MOI were dissolved into a serum-free medium and transfected for 4 h, respectively. Thereafter, the supernatant containing the adenovirus was abandoned and replaced with complete medium, followed by incubation for another 12 h. Successful transduction of the adenovirus was confirmed by the assessment of GFP expressions under fluorescent microscopy. The Ad-KDM3A-induced upregulation of the KDM3A protein was assessed by the Western blot assay.
9
Overexpression of CD9-GFP in HaCaT Cells
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Ad-CD9-GFP and CD9 mock vector Ad-GFP were purchased from Shanghai GeneChem, Co. Ltd. (Shanghai, China). Briefly, HaCaT cells inoculated into 6-wells plates, infected by Ad-CD9-GFP and CD9 mock vector Ad-GFP for 48h. Observe the transfection under fluorescence microscope and proved by Western blot. The molecular weight of CD9-GFP is about 55KD.
10
Notch3 Pathway Activation in Post-MI Rats
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Six- to eight-week-old male Sprague–Dawley rats (weighing 250 ± 20 g) were obtained from the Animal Research Center of Chongqing Medical University (Chongqing, China) and housed in a temperature-controlled environment on a 12-h/12-h light/dark cycle. All experimental procedures were approved by the Institutional Ethics Committee of Chongqing Medical University. The rats were anesthetized using sodium pentobarbital (60 mg/kg, i.p.), and thoracotomy was performed. We injected N3ICD (notch3 intracellular domain)-expressing adenovirus (Ad-N3ICD) and GFP-expressing adenovirus (Ad-GFP) [purchased from Genechem (Shanghai, China)] into the free anterior wall of the left ventricle at five different sites (2 × 109 pfu/ml, 5 μl per injection). Two days later, we introduced a myocardial infarction (MI) model as previously published (Qian et al., 2019 (link)). Briefly, ligation was accomplished at the proximal third of the left anterior descending (LAD) artery, and then the anterior wall of the left ventricle turned pale. Two months after thoracotomy, the rats were sacrificed, and the ventricular myocardium from the ischemic or region bordering the scar was harvested for further experiments.
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