Aliquots of human umbilical vein endothelial cells (HUVECs) (Yiyuan Biotechnology Corporation, GuangZhou, China) at a concentration of 2 × 104 cells per well were seeded onto Matrigel-coated wells (catalog number 356234; BD Corporation) of a 24-well plate. Tube formation assay was performed as reported previously [18 (link)]. The numbers of the vascular branches (formation of closed structures of HUVECs) were examined and calculated by randomly selecting five different fields per well in triplicate experiments by using a phase-contrast microscopy (CKX41, U-CTR30-2; OLYMPUS Japan) as described previously [5 (link), 18 (link)].
Matrigel coated wells
Manufactured by BD
Sourced in China
Matrigel-coated wells are specialized cell culture plates designed to provide a natural extracellular matrix environment for the growth and differentiation of various cell types. Matrigel is a reconstituted basement membrane extract derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, which mimics the complex structural and biochemical properties of the in vivo extracellular matrix. The Matrigel coating on the wells supports the attachment, proliferation, and differentiation of cells, making it a valuable tool for cell-based research and assays.
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6 protocols using matrigel coated wells
1
HUVEC Tube Formation Assay
2
Cell Migration and Invasion Assay
3
Vascular Network Formation Assay
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Aliquots of human umbilical vein endothelial cells (Yiyuan Biotechnology Corporation, Guangzhou, China) were seeded onto matrigel-coated wells (catalogue number 356234; BD Corporation, New York, USA) of a 24-well plate. After hypoxia/reoxygenation treatments, MSCs were then cocultured with human umbilical vein endothelial cells in 1% fetal calf serum-supplemented Dulbecco’s modified Eagle’s medium (catalogue number SH30021.01B; Hyclone, Logan City, Utah, USA). After incubating at 37°C for 6 hours, vascular network formation was examined by a phase-contrast microscopy (CKX41, U-CTR30-2; Olympus, Tokyo, Japan), and the number of the vascular branches was quantified by randomly selecting five fields per well as described previously [28 (link)].
4
Evaluating Cervical Cancer Cell Invasion
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Cervical cancer cell invasion was assessed by Boyden assay using transwell chambers (8 μm pore size; Costar, MA, USA). Chambers were coated with 100 μl matrigel and incubated overnight. Serum-starved SiHa (25,000) and HeLa (50,000) cells were seeded in the upper chambers of the Matrigel-coated wells (BD Biosciences, San Diego, CA, USA) containing 100 μl DMEM plus the desired treatment. Curcumin or emodin containing DMEM with or without TGF-β, supplemented with 10% FBS was placed in the lower chambers, and cells were allowed to migrate through the filter for 48 h. Cells that failed to migrate were removed from the upper surface of the chambers by scraping with a cotton swab. Cells that successfully migrated through the matrigel and the membrane barrier to the lower membrane were fixed in 100% methanol and stained with 2% crystal violet. Images of the stained cells were captured at 10X magnification. The extent of invasion was expressed as percentage of control deduced from the absorbance of crystal violet released by lysing the cells with 10% acetic acid at 595nm.
5
Angiogenesis Assay with Atox1 Knockdown
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HUVECs transfected with control siRNA or Atox1 siRNA were seeded on top of the thick growth factor-reduced Matrigel-coated wells (BD Biosciences) and incubated for 6 h at 37 °C. Images were taken with a Nikon digital camera, and eight random fields per well were analyzed.
6
Cell Migration and Invasion Assay
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Cells were serum-starved overnight and then 5×104 cells were plated in cell culture insert wells (BD Falcon) or Matrigel-coated wells (BD Biocoat) under the described conditions. After 24 or 48 hours, the unmigrated cells on the upper chamber were removed and inserts were fixed and stained with Diff-Quik. For co-culture experiments, CAF cells were seeded into the lower chamber and tumor cells into the upper chamber of cell culture inserts, as above. After 24 or 48 hours, the unmigrated cells on the upper chamber were removed and inserts were fixed and stained with Diff-Quik. Images were captured (Cool-Pix digital camera, Nikon) and the degree of migration or invasion was determined by the average number of migrated cells in five 100× fields (ImagePro, Media Cybernetics). Experiments were performed in triplicate and repeated three times. Measurements were taken at indicated time-points and were quantified with ImagePro. Differences between groups were analyzed by utilizing either conventional Student’s t test or ANOVA followed by post hoc comparisons based upon modified Newman-Keuls-Student procedure, where appropriate. Results are reported as mean +/− SEM. A p value of < 0.05 was considered significant and all are two-tailed.
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