The formation of tube-like structures was assessed in a Matrigel-coated 24-well plate (BD Biosciences, San Jose, CA) (Sharma et al., 2017 (link)). Briefly, human umbilical vein cells (HUVEC-1, ATCC PCS-100-010TM) were counted and seeded at a density of 20,000 cells/mm2 on reduced growth factor–containing Matrigel BD Biosciences, San Jose, CA #354230) with the addition of endothelial manipulation in aMSCs and nMSCs or conditioned medium from Empty a control. Cells were imaged after 6–12 h and a complete image of each well was reconstructed. The total tube length was then measured using ImageJ64, NIH (http://rsb.info.nih.gov/ij ).
Matrigel coated 24 well plate
Manufactured by BD
Sourced in United States
Matrigel-coated 24-well plates are a type of laboratory equipment used for cell culture applications. These plates provide a synthetic extracellular matrix (ECM) coating, which mimics the natural cellular environment and supports the growth and differentiation of various cell types.
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6 protocols using matrigel coated 24 well plate
1
Tube Formation Assay for Angiogenesis
2
Evaluation of hAEC-Secreted Cytokines' Pro-Angiogenic Potency
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The human umbilical vein endothelial cell line (hUVECs) is kindly provided by Dr. JufuTian (International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China), which is cultured in the DMEM/F12 supplied with 10% FBS. To assess the pro-angiogenic potency of hAEC-secreting cytokines, 5 × 104 hUVECs were cultured in the bottom chamber in Matrigel-coated 24-well plate (Matrigel, BD Biosciences, San Jose, CA, USA). 5 × 104 hAECs were cultured in the upper insert. And then, hUVECs were co-cultured with hAECs for 4 hours to allow the formation of tube-like structures. The images of tube-like structure were acquired under a light microscope, and the length of tube was measured using WimTube Quantitative Tube Formation Image Analysis Software.
3
Human OPC Derivation and Expansion
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Human oligodendrocyte progenitor cells (OPCs) were derived from NIH approved H9 embryonic stem cells were used (EMD Millipore; CS2044696). A total of 10,000 OPCs were suspended in 400 μL human OPC expansion complete media (human OPC basal medium, 2% neural supplement 1 [50X], 0.04% recombinant human bFGF, 0.04% OPC expansion supplement A [PDGF-AA], and 0.04% OPC expansion supplement B [NT3]; Millipore; SCM106) and grown in Matrigel-coated 24-well plates at 37 °C (BD; 356,234). Half of the cell culture medium was changed every 3 days until the cells were confluent.
4
HUVEC Tube Formation Assay
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Human umbilical vein endothelial cells (HUVECs) (Lonza) were grown in EGM-2 MV media. HUVECs, pretreated with BMP4 (2 nM) and BMP7v (100 ng/ml) were plated (70,000 cells/well) in Matrigel-coated 24 well plates (BD Biosciences), and incubated for 5 hours at 37°C. Tube formation was evaluated by phase-contrast microscopy, and cord length was measured using Image J analysis software (NIH).
5
In Vitro Endothelial Morphogenesis Assay
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In vitro endothelial cell morphogenesis assay was performed using Matrigel-coated 24-well plates (BD Biosciences, San Jose, CA, USA) (15 (link)). Briefly, 1×105 HMVEC-L cells were incubated with dDAVP or [V4Q5]dDAVP at a concentration of 100 nM. After allowing capillary tube formation for 24 h, randomly chosen fields were photographed at magnification ×100, and quantification was conducted. The number of capillary-like tubes formed in control cultures was taken as 100%.
6
Endothelial Progenitor Cell Angiogenesis
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EPCs were seeded at a density of 2×105 in Matrigel-coated 24-well plates (BD Biosciences) and incubated for 72 hours at 37℃. Tube formation in five randomly selected fields was examined under an Olympus light microscope (×400). The total tube length was determined.
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