Stk11fl/fl (#014143), Cdh5Cre (#006137), Cx3cr1Cre (#025524), Cx3cr1GFP (#005582), Gt(ROSA)26Sortm1(EYFP)Cos/J (#006148) mice were obtained from the Jackson Laboratory. VECad-CreERT2 mice were provided by Dr. Xin Zhang (University of Oklahoma Health Science Center). Stk11fl/fl, Cdh5Cre, and Stk11fl/flCdh5Cre (EC-specific Stk11-deleted, Stk11ec−/−) mice were generated as previously described38 (link). Stk11fl/flROSAEYFPCdh5cre mice were generated by crossing breeding Stk11fl/flCdh5Cre mice with Gt(ROSA)26Sortm1(EYFP)Cos/J reporter mice. Stk11fl/flCx3cr1Cre (DC-specific Stk11-deleted, Stk11dc−/−) were generated by crossing breeding Stk11fl/fl mice with Cx3cr1Cre mice. Stk11fl/flVECad-CreERT2 mice were generated by crossing breeding Stk11fl/fl mice with VECad-CreERT2 mice. Tamoxifen citrate (Sigma, # PHR2706) was intraperitoneally (i.p.) injected at 75 mg/kg body weight with corn oil (Sigma, #C8267) to either Stk11fl/flVECad-CreERT2 mice or littermate control mice at a concentration of 20 mg/mL every 24 h for a total of 5 consecutive days. Primers for genotyping are listed in Supplementary Table. 4 . Mice were housed in a controlled environment (20 ± 2 °C, 12-h/12-h light/dark cycle) in specific pathogen-free (SPF) animal facility. Experimental mice were co-housed with control mice. When euthanized, carbon dioxide inhalation chamber followed by cervical dislocation was performed. All animal protocols were approved by the Georgia State University Committee on the Use and Care of Animals (IACUC number: A18053).
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1
Conditional Genetic Manipulation of Endothelial and Dendritic Cells
2
Angiotensin II-Induced Hypertension in Mice
Male C57BL/6 mice were housed in the controlled environment with regulation of temperature (22 ± 1°C) and humidity (55%), and unrestricted access to food and water. All animal experiments were in accordance with the principles provided by the National Institute of Health Guideline and were approved by the Animal Care and Use Committee of Central South University (2019sydw0270). Male C57BL/6 mice (21–23 g) were treated with Ang II dissolved in sterile saline at 1 mg/kg/day using subcutaneous osmotic minipumps (Alzet, Cupertino, CA, United States) for 6 weeks (Zhang et al., 2014 (link)).
3
Isolation and Characterization of Mouse Vascular Cells
Mouse endothelial cells were isolated from adult C57BL/6J mouse lungs using collagenase I digestion (3 mg/mL in serum-free DMEM) at 37 °C in an incubator for 45 min, followed by CD31- and ICAM2-dynabeads-based double positive selection 45 (link), 46 (link). The isolated mouse lung endothelial cells were authenticated by endothelial marker VE-cadherin staining. More than 99% of cultured cells were VE-cadherin positive. The isolation of vascular smooth muscle cells (VSMCs) was performed using the explant method as previously described 47 (link), with minor modifications. VSMCs migrated from thoracic aorta explants at around 8 days, then, sprouted VSMCs were digested with 0.05% Trypsin-EDTA and reseeded for confocal microscopy or RNA isolation. More than 85% of cultured cells were smooth muscle alpha-actin (α-SMA) positive. Mouse peritoneal macrophages were isolated from the peritoneum cavity by injecting 5 mL ice-cold PBS/0.1% BSA 48 (link). Cells were cultured in complete media for 4 h, then, non-adherent cells were removed by washing. Macrophages were stained with CD68, and more than 99% of attached cells were CD68 positive.
4
Maternal Sex Hormone Effects on Metabolic Health
The animal protocol conformed to US NIH guidelines (Guide for the Care and Use of Laboratory Animals, No. 85–23, revised 1996), and was reviewed and approved by the Institutional Animal Care and Use Committee (from Wuhan University). The C57Bl/6 mice were housed 4 or 5 per cage on a 12:12-h light-dark cycle and were given phytoestrogen-free commercial rodent chow and water ad libitum on arrival.
Animal Protocol 1 : The 2-month female mice were anaesthetized by intraperitoneal injection of 100mg/kg ketamine/16mg/kg xylazine, and received treatments consisting of 60-day time release pellets (Innovative Research of America) that were implanted subcutaneously via a ~3mm incision on the dorsal aspect of the neck. Hormone pellets contained 5mg of either dihydrotestosterone (DHT, #A-161), estradiol (E2, #E-121), combined DHT and E2 (DHT/E2), or vehicle pellets (CTL) containing the same matrix but with no hormone [15 (link)]. After 1 week of surgery recovery, the female mice mated with male mice, and successful pregnancy was confirmed by examining the vaginal plugs. The pregnant mice were kept fed until baby delivery (~3 weeks) with another 3-week of lactation for the offspring. The mothers were sacrificed, and the blood was collected for analysis of hormones by radioimmunoassay. The EPCs were isolated from both bone marrow and peripheral blood [16 (link)] for gene expression analysis. The male offspring were selected and fed with high-fat diet (HFD, 60% calories from fat, Research Diets, #D12492) throughout the experiments. Parts of the young (4 months) and old (20 months) offspring were overnight-fasted, euthanized by 100mg/kg pentobarbital, and the EPCs were isolated for gene expression analysis. The EPCs numbers were counted [17 (link)] and the EPCs characteristics for colony form unit (CFU) [18 (link)] and migration [16 (link)] were evaluated. The tissues, including liver, heart and aorta were isolated for gene expression analysis or in vivo lipid uptake, and the blood was collected for measuring lipids, including total cholesterol, triglyceride, LDL and HDL cholesterol [14 (link)]. The MECs from the aorta were isolated for in vitro cell culture analysis [19 (link), 20 (link)], and the MECs from the thoracic aortas [21 (link)] were picked up by Laser Capture Microdissection (LCM) for mRNA analysis, and the vascular function was evaluated by vessel tension [22 (link), 23 (link)] and blood pressure [24 (link)–26 (link)] as described previously in our lab [14 (link)].
Animal Protocol 2 : The male offspring from CTL or DHT group in Animal Protocol 1 were used as recipients for bone marrow transplantation (BMT). Bone marrow cells were harvested from the tibias and femurs of the male offspring (4 months old) that were obtained from CTL group in Animal Protocol 1 as the donor for BMT. The isolated MNCs were purified by density centrifugation using Histopaque 1083® (#-1083-1, Sigma), and resuspended in 10ml of RPMI 1640 supplemented with 10% FBS and 2mM EDTA. We then added 4μg/ml of final concentration of polybrene with 100μl of concentrated lentivirus (1×107 cfu/ml), which included either Tie2-EMP, or Tie2-ERβ, or Tie2-SIRT1-C152(D) or Tie2-shERβ. We incubated the cells for 6 hours to achieve maximum 100% viral infection of cells. A parallel viral infection on the same MNCs cells was achieved using 100μl of 1×107 cfu/ml of pLVX-AcGFP1-C1 virus (#632155, Clontech) that can express GFP (green fluorescent proteins). The efficiency of viral infection was confirmed as ~100% using fluorescent microscopy. The above lentivirus infected MNCs were washed twice and resuspended by PBS, and then systemically transplanted (2×106 cells) into the above recipient male offspring (with CTL or DHT group) that had been lethally irradiated with 2 doses of 6 Gy 3 hours apart. All transplant-recipient mice were set aside for a minimum of 4 weeks to allow for complete reconstitution of the bone marrow [10 (link)], see details in S1 Table . The bone marrow transplanted mice were overnight-fasted and euthanized at the age of 20 months. The EPCs and MECs were isolated for in vitro cell culture analysis, or the MECs and SMCs (smooth muscle cells) were isolated by LCM for mRNA analysis, and parts of the mice were used for vascular function analysis.
5
Endothelial and Macrophage LKB1 Knockout Mice
6
Endothelial and Macrophage LKB1 Knockout Mice
The generation and characteristics of endothelium-specific LKB1-knockout mice (LKB1endo−/−) and macrophage-specific LKB1-knockout mice (LKB1macro−/−) were previously described.16 (link),30 (link) The animals were housed under specific pathogen-free conditions with a 12-hr light/dark cycle with food and water freely available. At least 5 mice in each group were used in the animal study. A neutralizing monoclonal anti-VEGF antibody (2G11-2A05, Bio-legend) or an isotype-matched control (RTK2758) was injected intravenously at 100 μg twice weekly, starting on the day of tumor implantation. All experiments were performed in accordance with the Animal Care and Use Committee of Georgia State University.
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