EAhy926 cells were seeded into a 6-well plate and cultured in full media until they reached confluency. The cells were serum starved for one hour, washed with PBS, and harvested. Matrigel (Corning Matrigel Growth Factor Reduced Basement Membrane Matrix; Corning, NY14831, USA) was diluted 1:1 with serum-free media and prewarmed at 37 °C in a 96 well plate. After seeding with approximately 85000 cells in serum free media on the Matrigel, the cells were incubated at 37 °C for 24 h in medium with DMSO ± PSP-2 at a final concentration of 5 µM. Two to three images per well were captured at 10x magnification, using a phase contrast inverted microscope (Axiovert-10, Carl Zeiss AG, Feldbach, Switzerland), equipped with a digital camera. Images were analyzed using the Angiogenesis Analyzer, a plugin developed for the ImageJ (Version 1.47t; NIH, Bethesda, MA, USA).[ref]
Matrigel
Manufactured by Zeiss
Sourced in Germany
Matrigel is a complex extracellular matrix preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma. It is commonly used as a basement membrane-like substrate for the culture of various cell types, including epithelial, endothelial, and stem cells.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (13 mentions)
Matrigel, by Corning (3 mentions)
Axio observer, by Zeiss (2 mentions)
Axiovision software, by Zeiss (2 mentions)
Observer z1 microscope, by Zeiss (2 mentions)
Microscopy, by Zeiss (2 mentions)
Axio imager, by Zeiss (2 mentions)
Axiovert 10, by Zeiss (1 mentions)
Matrigel growth factor reduced basement membrane matrix, by Corning (1 mentions)
Transwell insert, by Corning (1 mentions)
Cell observer microscope, by Zeiss (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Matrigel, by Merck Group (1 mentions)
Axiocam, by Zeiss (1 mentions)
Axiovert 40 cfl, by Zeiss (1 mentions)
Matrigel 354230, by Corning (1 mentions)
Axio microscope, by Zeiss (1 mentions)
Axio observer z1, by Zeiss (1 mentions)
Heparin, by Merck Group (1 mentions)
Fluorescent microscope, by Zeiss (1 mentions)
18 protocols using matrigel
1
Angiogenesis Assay with EAhy926 Cells
2
Transwell Invasion and Migration Assay
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The transwell invasion assay was performed using transwell inserts (Corning, NY, United States) and matrigel (BD Biosciences, San Jose, CA, United States). Briefly, the inserts with 8-µm pores were coated with 8 µL of matrigel. Then, 6 × 104 cells were suspended in 200 µL of serum-free medium and added to the upper compartment of the chamber. Subsequently, 600 µL of RPMI-1640 medium or DMEM containing 20% FBS was added to the lower compartment of the chamber as a chemo-attractant. After incubation for 24 h, cells that invaded through the matrigel were fixed with methanol, stained with 0.1% crystal violet, and photographed using a microscope (Zeiss, Axio Observer). The number of cells in five randomly selected fields from the central and peripheral regions of the filter was counted. The migration assay was conducted in a similar manner without matrigel coating.
3
Matrigel-Based Tube Formation Assay
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The neovascularization assays were performed in 24-well plates coated with Matrigel (Corning, NY). hCMEC/D3 cells (3 × 105) were plated on Matrigel and incubated for 18 h. Tube formation was observed at 5X using an inverted fluorescence Cell Observer microscope (Carl Zeiss, Jena, Germany). Length of tubes was measured in four independent fields using the “Angiogenesis Analyzer” software from Image J (National Institutes of Health).
4
Matrigel-based In Vitro Tube Formation Assay
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Ninety-six-well plates were coated with Matrigel (BD Biosciences, 354234) and incubated at 37°C for 30 min for solidification. Cells were plated onto Matrigel-coated wells at a density of 8 × 104 cells/ well with 100 μl αMEM without FBS and incubated at 37°C with 5% CO2 for 8 h. The images of tube formation were captured using a ZEISS fluorescence microscope and analyzed with NIH ImageJ software.
5
Mammosphere Formation Assay in Mice
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Cells extracted from 12 week virgin mice were resuspended in ice-cold Growth-factor-reduced Matrigel (BD Biosciences, CAT 356237). 8000 cells/well were seeded for primary Matrigel colony formation, 5000 cells/well were seeded for secondary Matrigel colony formation in 1 ml of DMEM/F12 with Pen/Strep and L-Glut supplemented with 20 ng/ml EGF (Sigma). Fresh EGF was added every 3–4 days. After 7 days, primary colonies were counted and dissociated enzymatically (10 min in TEG at 37 °C in water bath) and mechanically, and single cells re-seeded. qRT-PCR analysis and histologic analysis were performed as per mammospheres. Matrigel colonies were photographed with a bright field microscope and the colony size assessed using Axiovision software (Zeiss).
6
Angiogenic Tube Formation Assay
7
3D Differentiation Induction and Analysis
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To induce differentiation, cells were kept at 37ºC for at least 6 days without interferon-γ. After 1 week, the cells were used for 3D culture or other experiments. Growth factor–reduced Matrigel #354230 (Corning, Corning, NY) was used here, and cell dilutions were prepared so that there were approximately 6000 cells in 25 μL of medium; 25 μL of cell preparation was mixed with 50 μL of Matrigel, see Yanda et al.67 (link) Pictures were taken with a Zeiss Axio microscope (Zeiss, Oberkochen, Germany). Cystic areas were analyzed with ImageJ 1.50i (provided by the National Institutes of Health, Bethesda, MD).
8
In Vitro Tube Formation Assay
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The hLECs tube formation assay was performed by pipetting 200 μL Matrigel (BD Biosciences, Bedford, Massachusetts, USA) into each well of a 24-well plate firstly. After Matrigel polymerized for 30 min at 37 °C, hLECs (2 × 104) were added to each well with 200 μL of conditioned medium and incubated at 37 °C, 5% CO2 for 12 h. Images were photographed with ZEISS Axio Observer Z1 (ZEISS, Jena, Germany).
9
Angiogenic Potential of Cell Populations
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8-week-old C57BL/6J mice were used as recipients. Freshly isolated cell populations were resuspended in 0.4 ml Matrigel (Becton Dickinson) supplemented with 60 units/ml heparin (Sigma) and 150 ng/ml VEGF, and injected subcutaneously into the flank region. Matrigel plug was retrieved and analyzed 3-4 weeks later. Outgrowth were detected under fluorescent microscope (Zeiss) and categorized according to the total filled area of the Matrigel plug.
10
Transwell Invasion and Migration Assay
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The transwell invasion assay was done using the transwell (Corning, NY, United States) and matrigel (BD Biosciences, San Jose, CA, United States). Briefly, inserts with 8-μm pores were coated with 8 μL of matrigel. Cells (∼7.5 × 104) with 200 μL of serum-free medium were added into the upper compartment of the chamber. Subsequently, 600 μL of RPMI-1640 medium or DMEM containing 20% FBS was added into the lower chamber of the chamber as a chemo-attractant. After 36 h of incubation at 37°C in a humidified atmosphere containing 5% CO2, cells that migrated through the matrigel were fixed with methanol, stained with 0.1% crystal violet, and photographed using a microscope (Zeiss, Axio Observer). The cell number in five randomly selected fields from the central and peripheral regions of the filter was counted. The migration assay was conducted similarly without matrigel coating.
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