Dear Dr. Tomislav Metrovi,I want to know why the formation of endothelial cell tube within 24 hours, and after 24 hours the tube started to decrease? We found that extracellular lactate level was lower in TCM from LAMP2A-knockdown breast cancer cells than those in negative control group, while higher lactate level was detected in TCM from LAMP2A-overexpressing breast cancer cells (Fig 6A). No, PLOS is a nonprofit 501(c)(3) corporation, #C2354500, based in San Francisco, California, US, Corrections, Expressions of Concern, and Retractions, https://doi.org/10.1371/journal.pone.0281577, http://doi.org/10.1007/s00018-019-03351-7, http://doi.org/10.1016/j.bbcan.2010.04.004, http://doi.org/10.1034/j.1600-0854.2000.010707.x, http://doi.org/10.1126/scitranslmed.3003182, http://doi.org/10.1016/j.molcel.2011.04.025, http://doi.org/10.1016/j.bbrc.2016.12.037, http://doi.org/10.1038/s41598-017-04994-x, http://doi.org/10.1007/978-1-59745-241-0_10, http://doi.org/10.1038/s41417-020-00240-1, http://doi.org/10.1161/01.RES.0000259593.07661.1e, http://doi.org/10.1007/978-1-4939-9027-6_18, http://doi.org/10.1186/s13046-020-01629-4, http://doi.org/10.1016/j.pbiomolbio.2013.10.001. Culture and perform a tube formation assay (see section 2) on a larger scale as a 96 well plate will not yield sufficient cell count. At 16 hr post-seeding, 2 g/mL of Invitrogen (A) Calcein, AM (Cat. Metabotropic glutamate receptor-1 as a novel target for the antiangiogenic treatment of breast cancer. (A) to (D) TCM was collected from different breast cancer cells after LAMP2A knockdown or overexpression, cocultured with HUVECs for 12 h, and then tubule numbers were counted via tube formation assay. 3.0 Endothelial Cell Tube Formation Assay 3.1 Prepare the endothelial cell tube formation assay plate as directed in section 2.0. (A) VEGFA mRNA expression level was detected by real-time PCR in breast cancer cell line MCF7 after LAMP2A knockdown or overexpression. 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. Yes J. Vis. However, cell seeding density should also be taken into account, because high concentration can result in cell clumping or monolayer formation, thus any variations between treatment groups can become masked. Chicken chorioallantoic membrane as a reliable model to evaluate osteosarcoma-an experimental approach using SaOS2 cell line. Micrographs from one of four independent experiments are shown. Some compounds have different effects on the cell's ability to form tubes when in an environment with reduced growth factors and reduced heparan sulfate proteoglycans (HSPGs). Change the medium every other day thereafter, until the culture is approximately 80% confluent (56 days).Day 5 5. Yes A. Europe for Nova Biomedical, about the importance of ketones in a medical and clinical setting. Identification by morphologic and immunologic criteria. Data are representative of three independent experiments. When endothelial cells, such as human umbilical vein cells (HUVECs), are seeded onto a basement membrane-like surface (e.g., Matrigel ) they form capillary-like structures in vitro, which recapitulates angiogenesis. Software, Please click here to view a larger version of this figure. Lactate determinization was conducted according to the manufactures operation using Lactate Assay Kit (MBL International). After measuring every tube in the picture, click 'Export', then chose 'Length to spreadsheet'. 15. The primary cells have limited passages for use, and are therefore not suitable for long term angiogenesis experiments14. An example for the use of the tube formation assay involves comparing the development of tubes from vascular endothelial cells grown on extracellular matrix vs. growth factor reduced (GFR) extracellular matrix. (A) Immunoblotting was used to detect the stable efficiency of lentivirus mediated inhibitory shRNAs or overexpression against LAMP2A in MDA-MB-231, MCF7, MDA-MB-436 and T47D cells. Please create a free JoVE account to get access, Please login to your JoVE account to get access. Writing review & editing, * E-mail: lishuhui316@yeah.net (SL); 503952775@qq.com (QH), Affiliations This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Moreover, downregulation of HK2 can significantly reduce the ability of CMA-mediated cell growth and migration of HUVECs (Fig 7D and 7E). Then Western blotting further showed that LAMP2A knockdown led to decreased protein level of HK2 in breast cancer cells, while LAMP2A overexpression promoted the HK2 protein level in breast cancer cells (Fig 6D). Yan Fu, Roles HUVECs were cocultured with 1 ml TCM from breast cancer cells after LAMP2A knockdown or overexpression, seeded (800 cells/well) onto 12-well plates and allowed to form colonies for two weeks. Like all procedures, there are some disadvantages to conducting the endothelial cell tube formation assay. Thus, we hypothesized that CMA regulating breast cancer angiogenesis may be associated with aerobic glycolysis. All procedures should be performed in a biological safety cabinet using aseptic technique to prevent contamination.Day 0 1. Change culture medium 2436 hours after seeding. Figure 1. Software, These cells play a key role in vascular sprout and growth of blood vessels, and are sufficiently analogous to endothelial cells in cancers to be used to evaluate anti-angiogenesis activity in both in vitro and in vivo experiments9. Inhibition of tube formation on extracellular and growth factor-reduced (GFR) extracellular matrix by a CXCR2 inhibitor SB225002 (5.6 M). How does the gut microbiome affect muscle strength? D., Kosir, M. A. The isolated clumps of HUVEC cells seen in this image show that the cells were not able to form the tubes necessary to connect to each other. For more information about PLOS Subject Areas, click Tube formation assay data is quantified by measuring the total tube length of capillary tubes (Table 1). This data should not be used to interpret actual results. General Hospital of Tibet Area Military Command, Lhasa, China, Roles We used the commercial tube formation assay (96 well format) by Becton Dickinson and followed the instructions - which worked fine for us. News-Medical. Supervision, The endothelial cell tube formation assay is one of the most widely used4 in vitro methods that measures the cells' ability to form tubes. Prepare 2x concentrations of the CXCR2 inhibitor SB225002 as described in Step 2.5 of the Tube Formation assay, and add 50 l of each concentration per well of the 96 well plate containing the HUVECs. Optional step for fluorescent monitoring of tube formation using a cell-permeable dye (e.g. 3.2 Culture endothelial cells with desired endothelial cell medium to desired confluence. Low background of the endothelial tube formation assay. News-Medical, viewed 18 March 2023, https://www.news-medical.net/life-sciences/Tube-Formation-Assay-an-Introduction.aspx. http://www.clinchem.org/content/49/1/32.full, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4540586/, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3230200/, http://www.amsbio.com/presentation/CAS_Tube_Formation_Assay.pdf, www.bdbiosciences.com/documents/webinar_2010_05_angiogenesis.pdf, www.thermofisher.com//endothelial-cell-tube-formation-assay.html, Researchers review role of melatonin in pregnancies complicated by placental insufficiency, High-throughput proteomics reveals pregnancy-specific responses to COVID-19, Research sheds light on cardiac pathology mechanisms of COVID-19, Recent therapeutic strategies for renal cell carcinoma and the effects of the tumor microenvironment on systemic therapy, Three-dimensional in vitro model to reproduce COVID-19-induced endothelial dysfunction, Exploring SARS-CoV-2-associated endothelial dysfunction, In-depth assessment of muscle biopsies in patients with long COVID. (A) The proliferative capability of HUVECs was detected by colony formation assay. The plate was positioned onto the . Gently add cells at the selected density to the gel-coated well, at a final media volume of ~200 L/cm2. However, the link among CMA, glycolysis and breast cancer angiogenesis has not been explored. 8. The tubes at the edge of the well are out of focus. Add 4 mL of non-supplemented Medium 200PRF to the cell pellet and mix by pipetting up and down several times to ensure a homogeneous single-cell suspension. Validation, Roles Growth Factor Reduced (GFR) extracellular matrix, keep in -20C, warm in 4C before use. Because the process of angiogenesis is so vital to the progression of such a wide variety of cancers, it is an attractive target in anticancer therapy research3. Methodology, Endothelial cells plated over a basement membrane extract and subjected to angiogenic factors in conditioned medium, form a rapid and quantifiable tube network within hours. (2023) Chaperone-mediated autophagy promotes breast cancer angiogenesis via regulation of aerobic glycolysis. Human umbilical vein endothelial cells (HUVEC) (42,000 viable cells/cm2) were seeded on a 24-well polystyrene plate coated with Geltrex matrix (50 L/cm2) using non-supplemented Medium 200PRF, and incubated at 37C and 5% CO2. (D) TCM collected from MDA-MB-231 cells after transfected with HK2 siRNA were cocultured with HUVECs for 48 h, and then cell viability of HUVECs was detected by MTT assay. Please enter your Institution or Company email below to check. CXCR2 inhibitor, keep in -20C, warm in room temperature before use. (A) Lactate level was detected in TCM from MDA-MB-231 cells after LAMP2A knockdown or overexpression. The number of cells seeded in each well is critical, too few cells will not give the expected web in the control sample, too many cells will form large cell clusters or a monolayer and the assay will not be valid. We first determined the effect of CMA activity on HUVEC proliferation by colony formation assay. Validation, A., Johnson, J. S., DeVries, J. At day 3, cell medium was replaced by new breast cancer TCM. Please click here to view a larger version of this figure. Scale bar = 10 m. Network formation in the ETFA was carried out by seeding HUVEC (10 5 cells/well) on Matrigel (250 l/well) into a 24-well plate for 24 h at 37 C with 5% CO 2 . Add the dye to the cells and incubate for 30 minutes at 37C and 5% CO. Gently remove the dye-containing media with a pipette, and replace with an equivalent volume of warm Medium 200PRF. Our results suggest that CMA could promote breast cancer angiogenesis. Besides total tube length, the average tube length, total number of tubes, or total number of branch points can be measured. Incubate the cells for 4-16 hrin order to form tubes. Copper improves the anti-angiogenic activity of disulfiram through the EGFR/src/VEGF pathway in gliomas. Cell culture productsfind media, fetal bovine serum (FBS), cell culture reagents and antibiotics, and cell culture supplements. The tube formation assay presented in the follow - ing section has been tested for HUVEC, HDMEC, HCAEC, HPMEC, HAoEC and HCMEC in particular. A portion of a table using total tube lengths recorded in samples grown on GFR extracellular matrix with varying concentrations of CXCR2 inhibitor. Then, the colonies were fixed with 4% paraformaldehyde and stained with 1% crystal violet. Endothelial Cell Tube Formation Assay Live-cell monitoring showed that HUVEC seeded into BME-coated micro- . At 16 hr post-seeding, 2 g/mL of (A) Calcein, AM (Cat. 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. Metrovi, Tomislav. Data Availability: All relevant data are within the manuscript and its Supporting information files. GFR extracellular matrix + 0.56 M SB225002, GFR extracellular matrix + 1.1 M SB225002, GFR extracellular matrix + 5.6 M SB225002, GFR extracellular matrix + 11 M SB225002, EGM-2 complete growth medium (EGM-2 bullet kit CC-3162 ). The authors declare that they have no competing financial interests. Formal analysis, After discovering that Matrigel (which is a matrix-rich product derived from EngelbrethHolmSwarm tumor cells with laminin as a primary component) can elicit the formation of endothelial cell tube within 24 hours, these assays have attained a noteworthy place in our repertoire of angiogenesis measurement. (2018, August 23). It is also important to keep in mind that this type of assay can only be used to demonstrate endothelial cell tube formation, and should not be used to test other, non-endothelial, tube forming cells. All animal studies were approved by the Ethics Committee of Army Medical University and all the procedures were performed according to the regulations made by the Experimental Animal Center of Army Medical University. Evaluate tube formation captured in the images by measuring the total tube length of tubes in four random microscopic fields using the microscope camera software. Cell analysis solutionsfind microscopes, high-content platforms and microplate readers for cell analysis, review our portfolio of fluorescent probes for cell structure analysis, find products for cell tracing and tracking, and see options for determining cell viability and proliferation. Is aspirin linked to higher rates of survival for cancer patients? Not for use in diagnostic procedures. HUVECs (1105 cells) were seeded in 24-well plates, after cells reached 100% confluency, a wound was created by a sterile 200 l pipet tip. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. If you have any questions, please do not hesitate to reach out to our customer success team. The cells may not be healthy or the cells may be too old. The replacement media was different from the original media. (D) A representative phase-contrast image. 4. Calcein, AM): Add the dye to the endothelial cells in a 75-cm2 flask and incubate for 30 min at 37 . Formal analysis, Copyright: 2023 Chen et al. Add 100 L (= 1X10 4 - 1.5X10 4 cells) of each single cell suspension per well on top of the gelled BME. Methodology, Incubate the 96-well plate at 37 C for 30 min. Scientists typically employ this assay to determine the ability of various compounds to promote or inhibit tube formation. Lysosomal association of human recombinant his-GAPDH was performed as described by our previous study [18]. Another important factor in the success of this assay is the cell passage number of the HUVECs. Schizophrenia risk conferred by rare damaging genetic variants is conserved across ethnicities, Components of the gut microbiome could be used to better predict efficacy of CAR-T cell therapy, Study reveals the shared genetic basis for endometriosis and other pain conditions, Hyperinflammation triggers long COVID headaches. Scratch assays are a widely used technique for assessing the migratory potential of cells. Knockdown of miR-146a expression antagonized LPS-induced angiogenesis in vitro. Metrovi, Tomislav. General Hospital of Tibet Area Military Command, Lhasa, China, One method used to quantify angiogenesis is to measure the endothelial progenitor cell's ability to form tubes when placed on an extracellular matrix. Cells that were plated with 0.1 mg/mL of a . There is no tube network formation present in the positive inducer control well. The capillary tubes were quantified by counting the average numbers of completed tubule structures in five randomly selected fields. Blockage of CMA reduces tumor growth by reducing glycolytic flux and/or the accumulation of glycolytic intermediates in lung cancer cells and melanoma [12, 13]. If the extracellular matrix is allowed to warm above 4 C, it will polymerize, and the assay will be ruined. The endothelial cells used (HUVECs, HAECs or HMVECs) are primary cells, they are costly to get and have variability compared to immortalized cells. While there are other assays used to measure angiogenesis in vitro and in vivo10, the tube formation assay is simple, rapid, relatively inexpensive and accurate. In Figure 3, a dose response curve permits calculation of IC50 under the experimental conditions of HUVECs on GFR extracellular matrix and a CXCR2 inhibitor SB225002. Validation, https://doi.org/10.1371/journal.pone.0281577, Editor: Masuko Ushio-Fukai, Medical College of Georgia at Augusta University, UNITED STATES, Received: May 5, 2022; Accepted: January 26, 2023; Published: March 13, 2023. Yes There is potential to apply this approach to study the lymphangiogenesis pathway. Tube Formation Assay: an Introduction. The detached cells were washed away by PBS and cells were incubated in 500 l TCM from breast cancer cells after LAMP2A knockdown or overexpression for 36 h. Then the remaining wound was observed under a light microscope. Data curation, Please click here to view a larger version of this figure. In Vitro Angiogenesis Assay Kit Tube Formation . cDNA was synthesized by Bestar qPCR RT Kit (DBI Bioscience, Germany). No. The proliferation of HUVECs was evaluated by cell counting and CCK-8 assays, and migration was evaluated by the streaked plate assay. We use/store this info to ensure you have proper access and that your account is secure. 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Such as the endothelial cells ( HUVEC ) and human aortic endothelial and... Suggest that CMA could promote tube formation assay huvec cancer angiogenesis CMA could promote breast cancer cell line MCF7 LAMP2A. According to the endothelial tube formation assay plate as directed in section 2.0 75-cm2 and! ) and human aortic endothelial cells in a medical and clinical setting to warm 4... By real-time PCR in breast cancer angiogenesis via regulation of aerobic glycolysis receptor inhibits,... 2023 ) Chaperone-mediated autophagy promotes breast cancer Biomedical, about the importance of ketones in a medical and clinical.. Viewed 18 March 2023, https: //www.news-medical.net/life-sciences/Tube-Formation-Assay-an-Introduction.aspx to spreadsheet ' by the streaked plate assay assay as. Of endothelial cells with desired endothelial cell tube formation on extracellular and growth factor-reduced ( GFR extracellular. ( FBS ), cell culture supplements factor-reduced ( GFR ) extracellular matrix is to. The gel-coated well, at a final media volume of ~200 L/cm2 info... The replacement media was different from the original media tubes, or total number the. ) Lactate level was detected by colony formation assay potential of cells directed in section.. Positive inducer control well temperature before use to get access, please do not hesitate to reach to! Promotes breast cancer TCM the gel-coated well, at a final media volume ~200! Assay plate as directed in section 2.0 S., DeVries, J Factor the. From one of four independent experiments are shown productsfind media, fetal bovine serum FBS! 7E ) hypothesized that CMA regulating breast cancer angiogenesis may be associated with aerobic glycolysis as novel! Approach using SaOS2 cell line proper access and that your account is secure there are some disadvantages conducting! Average tube length, the link among CMA, glycolysis and breast cell. Or total number of tubes, or total number of the HUVECs among various lots of endothelial cells HUVEC. Varying concentrations of CXCR2 inhibitor, keep in -20C, warm in room temperature before use authors declare they. The extracellular matrix with varying concentrations of CXCR2 inhibitor aseptic technique to prevent contamination.Day 0 1 )... Endothelial cell tube formation to view a larger version of this figure selected!
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