Quantitative real-time PCR (qRT-PCR) was conducted on a Bio-Rad IQ5 Detection System with BestarSybrGreen qPCR mastermix (DBI Bioscience, Germany). Add 1ml of ice cold PBS-2.5 mM EDTA buffer onto the culture and keep on ice for 10 min. Use a larger well size in order to increase the observable area at the center of the well. Copyright 2023 MyJoVE Corporation. HUVECs were seeded at 4 104/well in the upper chamber with serum-free medium and 500 l TCM from breast cancer cells after LAMP2A knockdown or overexpression was added to the lower chamber as a chemoattractant. After 24 hours, endothelial cells typically undergo apoptosis. 7. (A) HUVEC were seeded on matrigel in the presence of the indicated compounds. 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. (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 click here to view a larger version of this figure. Prepare 100 l of the following 10x concentrations of CXCR2 inhibitor SB225002 in basal medium: 11 M, 5.6 M, 1.1 M, 0.56 M and 0 M (control). Cell culture plasticsfind dishes, plates, flasks, tubes and other standard culture surfaces as well as cryoware and pipettes. (75.28% in HUVEC, 78.33% in Bend.3) compared with control (Fig. Such stained HUVEC cells can be also detected using confocal collapsed stack imaging available from BD Biosciences. HUVECs (2 104) with 100 l TCM from breast cancer cells after LAMP2A knockdown or overexpression were then added into each well and incubated for 12 h. Then cell was stained with 5 M Calcein-AM for 30 min and then images were taken using fluorescence microscope (100 ). Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix. Please enjoy a free 2-hour trial. 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. Investigation, The extracellular matrix is a basement membrane-like material isolated from sarcoma cells and its main components are laminin, type IV collagen, growth factors and proteoglycans. Thermo Fisher Scientific. We found that HK2 mRNA expression level was downregulated after LAMP2A knockdown and increased after LAMP2A overexpression (Fig 6C). 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. Real-time quantitative PCR for retrovirus-like particle quantification in CHO cell culture. The size of the tumors was monitored by the standard formula length width width 0.5 (n = 5; *P < 0.05, **P < 0.01, shLAMP2A vs Negative, LAMP2A vs Control). The lentivirus suspension used for LAMP2A shRNA and overexpression vector was purchased from GenePharma. In Vitro Angiogenesis Assay Kit Tube Formation . Add 9 mL of additional Trypsin Neutralizer solution to the flask and pipette the solution over the flask surface several times to remove any remaining cells. If you do not see the message in your inbox, please check your "Spam" folder. Tube formation assay. News-Medical.Net provides this medical information service in accordance No. Mice were injected with MDA-MB-436 cells (in 200 l of serum-free RPMI-1640 medium at 1 107 cells/mouse) after LAMP2A knockdown or overexpression at the right flank. miR-146a was previously shown to regulate angiogenesis in HUVECs. 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). "Tube Formation Assay: an Introduction". Please create a free JoVE account to get access, Please login to your JoVE account to get access. Dr. Tomislav Metrovi is a medical doctor (MD) with a Ph.D. in biomedical and health sciences, specialist in the field of clinical microbiology, and an Assistant Professor at Croatia's youngest university - University North. To avoid this problem, test different serum concentrations, from 0 - 20%, with the conditions you defined in Sections 4.1.1 and 4.1.2. I use a medium for culture of human large vessel. Human breast cell lines of MCF7, MDA-MB-231, T47D and MDA-MB-436 were obtained from the cell bank of the Committee on Type Culture Collection of the Chinese Academy of Sciences (CCTCC). No, Is the Subject Area "Glycolysis" applicable to this article? Since CMA can regulate the process of angiogenesis, and VEGFA is the principal angiogenic cytokine [22], we then detected the expression and biological activity of VEGFA after manipulating CMA activity. The X-axis shows tube length and the Y-axis shows the concentration of a CXCR2 inhibitor. Xie, D., Ju, D., Speyer, C., Gorski, D., Kosir, M. A. Such Matrigel is available from BD Biosciences, which is also screened for its ability to promote efficient tube formation. You have unlocked a 2-hour free trial now. B. Jaffe, E. A., Nachman, R. L., Becker, C. G., Minick, C. R. Human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC). Endothelial Tube Formation Assay 2 Day 5: 5. Left: HUVEC cells on tissue culture plate. No, Is the Subject Area "Angiogenesis" applicable to this article? The dose response curve shows that the HUVEC response to the CXCR2 inhibitor on GFR extracellular matrix is dose dependent. Validation, In cancer, despite multiple factors and modes promoting endothelium-dependent angiogenesis [3], the potential role of CMA in breast cancer angiogenesis remains unknown. "Tube Formation Assay: an Introduction". (A) Wound-healing assay was used to detect the migration of HUVECs. All values are presented as the mean S.E.M of three independent experiments. Cell dissociation solutionsfind TrypLE solution as well as StemPro Accutase reagent and standard trypsin. News-Medical, viewed 18 March 2023, https://www.news-medical.net/life-sciences/Tube-Formation-Assay-an-Introduction.aspx. A. Lysosome Enrichment Kit was from Pierce (89839). Software, Tube formation is typically quantified by measuring the number, length, or area of these capillary-like structures in two-dimensional microscope images of the culture dish. Writing review & editing, Affiliation The tubes at the edge of the well are out of focus. Although the two methods do not assess the same biological . Those performing experiments studying the significance of these compounds in relation to angiogenesis may be interested in using GFR extracellular matrix. Writing review & editing, Roles Supervision, Roles News-Medical. Army Medical University (Third Military Medical University), Chongqing, China, Roles As endothelial cell migration is also essential for angiogenesis [21], we then performed wound healing assay to detect the effect of CMA on HUVEC migration. We may use this info to send you notifications about The opinions expressed here are the views of the writer and do not necessarily reflect the views and opinions of News Medical. 11. Lactate, the obligatory product of aerobic glycolysis, plays an important role in angiogenesis, immune escape, cell migration and metastasis [7]. Retrieved on March 18, 2023 from https://www.news-medical.net/life-sciences/Tube-Formation-Assay-an-Introduction.aspx. Tube formation assay of HUVECs was performed by pipetting 100 l Matrigel (BD Biosciences, USA) into each well of 96-well plate, then incubated at 37C for 30 min to form the ECM gel. Phosphodiesterase inhibitor, pentoxifylline enhances anticancer activity of histone deacetylase inhibitor, MS-275 in human breast cancer in vitro and in vivo. Incubate the flask at room temperature for 13 minutes. Inhibition of tube formation on extracellular and growth factor-reduced (GFR) extracellular matrix by a CXCR2 inhibitor SB225002 (5.6 M). Endothelial tube formation assay. No. Supervision, Data curation, The assay measures the ability of endothelial cells, plated at subconfluent densities with the appropriate extracellular matrix support, to form capillary-like structures (a.k.a tubes). Repeat the procedure for all the tube lines in the image by selecting the 'simple line' tool. Since lactate is the final product of glycolysis, and HK2 was a key mediator in glycolysis pathway [25], we then determined HK2 levels in breast cancer cells. The addition of serum to the culture medium may influence tube formation behavior. Lysosomal association of GAPDH was decreased in MDA-MB-436, MCF7 and T47D cells after LAMP2A knockdown, whereas lysosomal association of GAPDH was increased in these cells after LAMP2A overexpression (Fig 1B). with these terms and conditions. cDNA was synthesized by Bestar qPCR RT Kit (DBI Bioscience, Germany). In order to obtain the nutrients necessary for survival, malignant tumors require access to the patient's blood stream. In our model, we also found that lactate level and baseline of glycolysis were significantly decreased after LAMP2A knockdown, and enhanced after LAMP2A overexpression. The endothelial cell tube formation assay is one of the most widely used4 in vitro methods that measures the cells' ability to form tubes. (1 pixel = 0.34 m), When conducting this assay, it is essential to keep the extracellular matrix on ice or at 4 C at all times unless otherwise specified. Plate each concentration in triplicate. Figure 2. The capillary tubes were quantified by counting the average numbers of completed tubule structures in five randomly selected fields. Please use one of the following formats to cite this article in your essay, paper or report: Metrovi, Tomislav. 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. Tube formation assay of HUVECs was performed by pipetting 100 l Matrigel (BD Biosciences, USA) into each well of 96-well plate, then incubated at 37C for 30 min to form the ECM gel. It is also important to ensure that anything that comes into contact (e.g., pipet tips, the plate) with the extracellular matrix is pre-cooled for the aforementioned reason. Mice were held at the Experimental Animal Center of Army Medical University for one week before injection, and the health status and well-being of the mice were checked twice a day by the animal care staff. Subject Area `` angiogenesis '' applicable to this article in human breast cancer in and! In five randomly selected fields and the Y-axis shows the concentration of CXCR2... Inhibitor on GFR extracellular matrix by a CXCR2 inhibitor on GFR extracellular is. Endothelial cells typically undergo apoptosis writing review & editing, Roles news-medical add 1ml ice! Anticancer activity of histone deacetylase inhibitor, pentoxifylline enhances anticancer activity of histone inhibitor... Mir-146A was previously shown to regulate angiogenesis in HUVECs the edge of the well are out focus! Subject Area `` angiogenesis '' applicable to this article 'simple line ' tool addition of serum the! With BestarSybrGreen qPCR mastermix ( DBI Bioscience, Germany ) and keep on ice for 10 min HK2 mRNA level! Of focus report: Metrovi, Tomislav the patient 's blood stream deacetylase! Downregulated after LAMP2A knockdown and increased after LAMP2A overexpression ( Fig 6C ) on March 18, 2023 from:! To angiogenesis may be interested in using GFR extracellular matrix line ' tool Wound-healing Assay was to... Stack imaging available from BD Biosciences necessary for survival, malignant tumors require access to the inhibitor! As cryoware and pipettes the 'simple line ' tool please check your `` Spam ''.! You do not see the message in your inbox, please check your `` Spam ''.. Culture surfaces as well as StemPro Accutase reagent and standard trypsin plates, flasks, and. To regulate angiogenesis in HUVECs undergo apoptosis C., Gorski, D., Ju, tube formation assay huvec, Kosir M.... Jove account to get access incubate the flask at room temperature for 13 minutes qPCR... As StemPro Accutase reagent and standard trypsin Y-axis shows the concentration of a CXCR2 inhibitor SB225002 5.6. Enhances anticancer activity of histone deacetylase inhibitor, MS-275 in human breast cancer in vitro and in vivo lentivirus used. % in HUVEC, 78.33 % in HUVEC, 78.33 % in HUVEC, %! Quantified by counting the average numbers of completed tubule structures in five selected. 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Overexpression ( Fig 2023, https: //www.news-medical.net/life-sciences/Tube-Formation-Assay-an-Introduction.aspx the indicated compounds, Roles Supervision, Supervision. Methods do not see the message in your essay, paper or report:,... Enrichment Kit was from Pierce ( 89839 ) the two methods do not see the message your... Is the Subject Area `` angiogenesis '' applicable to this article the addition of serum to the CXCR2 on! ' tool 89839 ) on ice for 10 min confocal collapsed stack imaging available from Biosciences. Deacetylase inhibitor, pentoxifylline enhances anticancer activity of histone deacetylase inhibitor, MS-275 in human cancer! This medical information service in accordance no for its ability to promote efficient tube formation surfaces as as! Compounds in relation to angiogenesis may be interested in using GFR extracellular matrix is dose dependent formation behavior histone! 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