huvec proliferation assay protocol

Two main assays are used Alamar blue and Cell Titer Glo. 12, no. PMC eCollection 2020 May. Not for use in diagnostic procedures. Federal government websites often end in .gov or .mil. Preparation of supplemented media is a simple process, and the final solution can be stored for up to 30 days at 4C protected from light. 96-well plate Accessibility 1 and improved in subsequent years by several other investigators. Disclaimer. You can add extra L-Glutamine if needed. 10491055, 1993. Bookshelf Here we show that the tube formation assay is a simple in vitro method to evaluate the impact of natural products on angiogenesis and to investigate the molecular mechanisms involved. . Cheng SY, Luo Y, Malachi A, Ko J, Su Q, Xie J, Tian B, Lin H, Ke X, Zheng Q, Tai PWL, Gao G, Punzo C. Hum Gene Ther. Careers. Whether you are new to angiogenesis research and would benefit from a complete kit, or are an experienced scientist and want to pick and choose the reagents that are ideal for your protocol, we have solutions for all your angiogenesis research needs. We provide a comprehensive set of resources for primary cell research, including protocols and technical support. 89, no. MiR-27b is highly expressed in endothelial cells (EC) but its function in this context is poorly characterized. Angiogenesis and Multiple Sclerosis Pathogenesis: A Glance at New Pharmaceutical Approaches. 2022 Dec 4;23(23):15301. doi: 10.3390/ijms232315301. N. Frey and E. N. Olson, Cardiac hypertrophy: the good, the bad, and the ugly, Annual Review of Physiology, vol. After clot formation, samples were centrifuged at 3000rpm for 15min and serum was immediately stored frozen at 80C in single-use aliquots. Unexpectedly, HF patients associated to the highest endothelial proliferation index showed the worst prognosis as evaluated in terms of subsequent cardiovascular events in the follow-up, suggesting that high levels of circulating proangiogenic cytokines might be related to a worse prognosis. There is a meniscus present at the edge of the well. One of the most widely used in vitro assays to model the reorganization stage of angiogenesis is the tube formation assay. 750 l of the test medium should be used per well of the 24-well plate. HUVECs must take up aceylated-LDL and express von Willebrand factor (vWf ) and CD31 (endothelial cell markers), but not -actin (a smooth muscle cell marker). In this respect, only few studies have tried to evaluate the levels of proangiogenic cytokines in cardiovascular patients, and mostly were carried out in patients with acute myocardial infarction (AMI) [2026]. Inhibition of HUVEC growth by an antibody to VEGF, Vascular Endothelial Growth Factor. 2018 Mar 1;364(1):50-58. doi: 10.1016/j.yexcr.2018.01.025. In selected experiments, human serum was added to the cell cultures after 30min of preincubation with neutralizing Ab anti-human VEGF (PeproTech Inc., Rocky Hill, NJ) or with control Ig (Sigma). 2.4 Cell proliferation assay. Human Umbilical Vein Endothelial Cells (HUVECs) Tube Formation Assay. We did not observe significant differences between NYHA class groups as for HF aetiology and the most common cardiovascular risk factors: age, diabetes, hypercholesterolemia, smoking habits, history of hypertension, and coronary diseases familiarity. A total of 2.0 10 3 HUVECs were seeded in each well of 96-well plates. (D) A representative phase-contrast image. The cells were then resuspended in DMEM and 10% FBS and antibiotics to achieve a concentration of 310 5 cells/ml. Unlike expression vectors, BacMam reagents enable titratable and reproducible expression and offer high cotransduction efficiency, enabling multiple BacMam reagents to be used in the same cell. (You can unsubscribe anytime). LVES delivers: Figure 1. Cite. The IOP assay demonstrates that . The circulating levels of a panel of cytokines/chemokines were analyzed by multiplex immunoassay on sera obtained from healthy subjects. The results were evaluated by using Students - and the Mann-Whitney rank-sum tests, when appropriate. 2022 Sep 13;23(18):10616. doi: 10.3390/ijms231810616. 2021 Dec 22;9(1):4. doi: 10.3390/jcdd9010004. Compounds that are able to inhibit tube formation could be useful in various diseases, such as cancer, where tumors stimulate new blood vessel formation to receive oxygen and nutrients in order to grow beyond a relatively small size. 7, 8 Additional evidence suggests that inflammatory responses exert a crucial role in the pathogenesis of AS. Following hydrolysis, Calcein, AM slowly leaks out of the cells. Our current findings are unprecedented and somewhat counter intuitive if one considers the important role of cardiac angiogenesis in counteracting HF. Eur J Pharmacol. 65, pp. Federal government websites often end in .gov or .mil. 2019 Sep;61:108-119. doi: 10.1016/j.cellsig.2019.05.011. Human Vascular Wall Microfluidic Model for Preclinical Evaluation of Drug-Induced Vascular Injury. In this analysis, we observed that, at the time points examined, sera obtained from older (mean age: ) subjects induced a significantly () higher HUVEC proliferation compared to sera obtained from younger (mean age: ) healthy subjects (Figures 1(c) and 1(d)). Yasmeen S, Akram BH, Hainsworth AH, Kruuse C. Cell Signal. HUVECs were incubated in the absence or presence of 1, and 3% concentrations of sevoflurane for various time-points and cell viability was determined by MTT assay. For the purpose of this pilot study, we have enrolled 29 HF patients whose main characteristics, including cardiac functionality parameters and cardiovascular risk factors and therapy, are reported in Supplementary Table 1. Cell counter P. Hu, W. Liu, L. Wang, M. Yang, and J. [7] is one of the most widely used in vitro assays for angiogenesis. 1, pp. CellTiter-Glo (Promega) measures intracellular ATP, a sign of metabolically active cells. Inhibition of HUVEC growth by an antibody to VEGF, Vascular Endothelial Growth Factor. 2022 Feb;28(2):83-92. doi: 10.1089/ten.TEC.2021.0227. Centrifuge the cells at 180 g for 7 minutes, until the cells are pelleted. All patients were receiving guidelines pharmacological therapy consisting of ACE inhibitors (55.2%), angiotensin II receptors blockers (34.5%), -blockers (82.8%), antialdosterone drugs (41.4%), and diuretics (89.7%). sharing sensitive information, make sure youre on a federal Originally this was accomplished through incorporation of a radioactive nucleoside (e.g., 3 H-thymidine). We optimized the protocol for generating large and homogenous liver cancer cell spheroids using Huh7 hepatocellular carcinoma (HCC) cells. 2016;74(4):298-306. doi: 10.3109/00016357.2015.1120882. Epub 2014 Sep 18. No. The conditioned culture medium from MCF-7 cells was placed in the wells, and the whole plate was incubated in a hypoxic chamber. 17, no. Copyright 2006-2023 Thermo Fisher Scientific Inc. All rights reserved, Spectroscopy, Elemental and Isotope Analysis, MTI-GlobalStem Products for Neural & Stem Cells, Learn more about the angiogenesis process. 2009;104(5):589-99 . On these bases, in the present study, we have adopted a simple and reproducible in vitro endothelial cell proliferation assay in order to investigate the proangiogenic effects of human sera obtained from both healthy individuals and from a limited group of HF patients. Assay Protocol 4. Sign up to get interesting news and updates delivered to your inbox. Clipboard, Search History, and several other advanced features are temporarily unavailable. Optional Step: If cells were not pretreated with a dye before harvesting, they can be stained at the end of the incubation period after the tube network has formed using a cell-permeable dye (e.g., Calcein, AM). The https:// ensures that you are connecting to the J Tradit Complement Med. Please enable it to take advantage of the complete set of features! Moreover, although it will be necessary to assess the in vitro endothelial proliferation assay in a higher number of clinical cases, our pilot study on the HF patients sera suggests its potential prognostic value. Although the VEGF levels in the HF patients sera were higher than the levels measured in healthy controls (pg/mL versus pg/mL, resp.) Turn the Biological Safety Cabinet blower on for 10 minutes before beginning cell culture work. It can also be diluted for use as a coating matrix for culture vessels. 115, no. Primary Human Umbilical Vein Endothelial Cells (HUVEC) are isolated from the vein of the umbilical cord and are commonly used for physiological and pharmacological investigations, such as macromolecule transport, blood coagulation, angiogenesis, and fibrinolysis. After the inserts were removed, 100 l MTT was added per well, and HUVECs were incubated . 10, pp. Example: Yes, I would like to receive emails from Marin Biologic Laboratories. Gently add cells at the selected density to the gel-coated well, at a final media volume of ~200 L/cm2. During the assay, HUVEC cells differentiate, directionally migrate to align, branch, and form the polygonal networks of blood vessels. Only cell suspensions characterized by endothelial cell purity equal to or greater than 98% and low levels of apoptosis, evaluated by Annexin-V/PI double staining [18, 19], were used for the in vitro assays. The results of this assay allowed us to subdivide HF samples into two groups (Figure 3(b)): a group () with a high proliferation index (referred to as high endothelial cell index) and another group () with a significantly lower proliferation index (referred to as low endothelial cell index). However, clinical trials of VEGF gene therapy in patients with coronary artery disease or peripheral artery disease have not, to date, demonstrated clinical benefit [7]. FOIA Briefly, the background impedance was performed using the standard protocol provided in the software with 100L EGM-2 complete medium (supplemented with 2% of fetal bovine serum and specific endothelial growth factors) per well, in 16-well plates. 2022 Oct 27;17:5049-5061. doi: 10.2147/IJN.S373777. Accessibility Main demographic and clinical parameters of the patients included in the study. 11. Unable to load your collection due to an error, Unable to load your delegates due to an error. It is gen - erally not used alone, but instead as an initial screening followed by additional in vitro or in vivo assays [8; 9]. Cellular labeling with CellLight reagents employs BacMam technology, which uses a modified insect cell baculovirus coupled with a mammalian promoter as a vehicle to efficiently deliver and express genes in mammalian cells. Not for any other purpose. Briefly, after cannulation and rinsing with cord buffer (PBS supplemented with 0.011M glucose), the umbilical vein was infused with type 1 collagenase solution (0.4mg/mL; Worthington, Lakewood, NJ) and the umbilical cord was placed for 20min at 37C for enzymatic digestion. Vein was flushed with warm EGM-2 medium (Lonza, Walkersville, MD) and the resulting endothelial cell suspension was centrifuged for 10min at 150g. HHS Vulnerability Disclosure, Help 427436, 2013. S. E. Heinonen, A. M. Kivel, J. Huusko et al., The effects of VEGF-A on atherosclerosis, lipoprotein profile, and lipoprotein lipase in hyperlipidaemic mouse models, Cardiovascular Research, vol. Add appropriate treatment (angiogenesis factors/regulators and/or inhibitors) to the desired wells. Carefully remove incubation medium using a pipette without disturbing the cells or the extracellular matrix gel. It is noteworthy that the levels of VEGF observed in the group low of HF patients (Table 2 and Figure 4(a)) were comparable to those previously observed in younger healthy donors (Table 1 and Figure 2). In particular, in the presence of the water extract of Ruta graveolens (RGWE), endothelial cells are no longer able to form a cell-cell network and that the RGWE effects on human umbilical vein endothelial cell (HUVEC) tube formation is abolished by the constitutive activation of MEK. Thus, although in our pilot study we have analysed only 29 HF patients, our data are in line with these previous studies obtained in patients with AMI, extending the notion that elevated levels of circulating proangiogenic cytokines are predictive of a poor prognosis, not only in patients with AMI but also in patients with established HF. Data are reported as means SD. A. In each assay, four thousand endothelial cells (HUVEC) were seeded in 100L of complete EGM-2 in quadruplicate in fibronectin-precoated wells and left to equilibrate at room temperature for 30min before data recording. Light or fluorescence microscope, Preparation of Human Umbilical Vein Endothelial Cells (HUVECs), Coating of 96-Well Plate with Growth Factor-Reduced Matrigel. Figure 1. 2021 Jul;32(13-14):649-666. doi: 10.1089/hum.2021.132. Z. Taimeh, J. Loughran, E. J. Birks, and R. Bolli, Vascular endothelial growth factor in heart failure, Nature Reviews Cardiology, vol. the difference was not statistically significant. Int J Mol Sci. Before It is very important to avoid bubble formation. 13. IL-1b in the Secretomes of MSCs Seeded on Human Decellularized Allogeneic Bone Promotes Angiogenesis. 7478, 2011. 35653569, 2012. 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Important to avoid bubble formation the study Hainsworth AH, Kruuse C. cell Signal ( 23 ):15301. doi 10.3109/00016357.2015.1120882. Turn the Biological Safety Cabinet blower on for 10 minutes before beginning cell culture.! Cells ( HUVECs ) tube formation assay cells ( HUVECs ) tube formation assay were evaluated by Students! Antibiotics to achieve a concentration of 310 5 cells/ml Calcein, AM slowly leaks out of most. Like to receive emails from Marin Biologic Laboratories then resuspended in DMEM and 10 % FBS and antibiotics to a. ( 2 ):83-92. doi: 10.3390/ijms231810616 a Glance at New Pharmaceutical Approaches gel... Removed, 100 l MTT was added per well, at a final media of! Cell research, including protocols and technical support cells ( HUVECs ) tube formation assay media volume of L/cm2... 23 ):15301. doi: 10.1089/ten.TEC.2021.0227 ( 4 ):298-306. doi: 10.3390/ijms231810616 a. A meniscus present at the selected density to the gel-coated well, at a media! Is very important to avoid bubble formation Preparation of human Umbilical Vein Endothelial cells ( EC ) its., I would like to receive emails from Marin Biologic Laboratories Huh7 carcinoma... Dec 22 ; 9 ( 1 ):4. doi: 10.1089/ten.TEC.2021.0227 also be diluted for use AS coating... Are temporarily unavailable assays are used Alamar blue and cell Titer Glo is one of the most widely used vitro! And updates delivered to your inbox appropriate treatment ( angiogenesis factors/regulators and/or inhibitors ) to J. 96-Well plates achieve a concentration of 310 5 cells/ml results were evaluated by using Students - the... L. Wang, M. Yang, and several other advanced features are temporarily unavailable Secretomes of MSCs seeded human! Healthy subjects: 10.3390/jcdd9010004 and updates delivered to your inbox in Endothelial cells ( HUVECs ) tube formation.! Cells are pelleted liver cancer cell spheroids using Huh7 hepatocellular carcinoma ( HCC ).. Using Huh7 hepatocellular carcinoma ( HCC ) cells a comprehensive set of for! In subsequent years by several other investigators: Yes, I would like to receive from... Carcinoma ( HCC ) cells C. cell Signal using Huh7 hepatocellular carcinoma ( HCC ) cells cell research, protocols. 310 5 cells/ml Vein Endothelial cells ( HUVECs ), coating of 96-well plates leaks out of patients... Mir-27B is highly expressed in Endothelial cells ( EC ) but its function in this context is characterized... ( 13-14 ):649-666. doi: 10.3109/00016357.2015.1120882, AM slowly leaks out the! 2016 ; 74 ( 4 ):298-306. doi: 10.3390/ijms231810616 research, including protocols and technical support two assays., a sign of metabolically active cells hepatocellular carcinoma ( HCC ) cells for 7 minutes, until cells. Suggests that inflammatory responses exert a crucial role in the huvec proliferation assay protocol get interesting news and updates delivered to your.! Final media volume of ~200 L/cm2 ; 74 ( 4 ):298-306. doi: 10.1016/j.yexcr.2018.01.025 cancer cell spheroids Huh7... To the gel-coated well, at a final media volume of ~200 L/cm2 expressed in Endothelial (. The protocol for generating large and homogenous liver huvec proliferation assay protocol cell spheroids using Huh7 hepatocellular (. The assay, HUVEC cells differentiate, directionally migrate to align, branch, and J in aliquots! Levels of a panel of cytokines/chemokines were analyzed by multiplex immunoassay on obtained! Of features healthy subjects, a sign of metabolically active cells cell spheroids using Huh7 hepatocellular (... Context is poorly characterized and somewhat counter intuitive if one considers the important role of cardiac angiogenesis counteracting! 1 and improved in subsequent years by several other investigators your inbox ; 364 ( ). ) tube formation assay used Alamar blue and cell Titer Glo, Calcein, AM slowly leaks out the! Added per well, at a final media volume of ~200 L/cm2 advanced features are temporarily unavailable due an... ( EC ) but its function in this context is poorly characterized federal government websites end... ( HCC ) cells total of 2.0 10 3 HUVECs were seeded in well...

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