After thawing Matrigel (EMD Millipore, Billerica, MA, USA) on ice the 96-well plate coated with 50 μL Matrigel in each well was then incubated at 37°C for 30-min to allow the Matrigel to polymerize. To examine the effect of p53 phenotype on tumor cell-induced tube formation of HUVECs (ATCC), a conditioned medium was collected from R72wt or mutant phenotypes transfected Caco2 cells as indicated and used as the growth medium for HUVECs. A total of 1 × 104 HUVECs were seeded into each well that had conditioned medium. Cells were then incubated for 8 h to allow the formation of tube-like structures. There are three parameters by which capillary structure formation can be considered: capillary length, number of capillaries or branched tubes.
Matrigel
Manufactured by Merck Group
Sourced in United States, Germany, China, United Kingdom, Macao, Sao Tome and Principe, Japan, France, India, Canada, Denmark, Italy
Matrigel is a complex mixture of extracellular matrix proteins and growth factors derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. It is commonly used as a substrate for the culture of various cell types, including epithelial, endothelial, and stem cells. Matrigel provides a physiologically relevant environment that supports cell attachment, growth, and differentiation.
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Lab products found in correlation
Crystal violet, by Merck Group (117 mentions)
Matrigel, by BD (97 mentions)
Transwell chamber, by Corning (58 mentions)
Matrigel, by Corning (56 mentions)
Transwell chamber, by Merck Group (36 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (25 mentions)
Inverted microscope, by Olympus (25 mentions)
Fibronectin, by Merck Group (23 mentions)
Ix71 inverted microscope, by Olympus (23 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (21 mentions)
Paraformaldehyde (pfa), by Merck Group (20 mentions)
Transwell plate, by Corning (16 mentions)
Triton x 100, by Merck Group (16 mentions)
Transwell chamber, by BD (15 mentions)
Upper chamber, by Corning (13 mentions)
Crystal violet, by Beyotime (13 mentions)
Dmem f12, by Thermo Fisher Scientific (13 mentions)
Transwell insert, by Corning (12 mentions)
Streptomycin, by Thermo Fisher Scientific (12 mentions)
Transwell migration chambers, by Merck Group (12 mentions)
1 099 protocols using matrigel
1
Endothelial Tube Formation by Tumor Cells
2
Transwell Migration Assay Protocol
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Matrigel (EMD Millipore, Merck KGaA) was equilibrated overnight to a liquid state, and 200 μL Matrigel was supplemented with 200 μL serum-free medium, and then mixed well to dilute Matrigel. Next, 50 μL Matrigel was added to each Transwell plate, and incubated for 2–3 h to insure a solid state. A cell suspension was prepared using medium containing 20% FBS, 200 μL cell suspension was added to the apical chamber of each well, and 800 μL conditioned medium containing 20% FBS was added to the basolateral chamber. The transwell plate was then rinsed with formaldehyde for 10 min, washed three times with water, and stained with 0.1% crystal violet staining solution. The cells on the upper surface were wiped away with cotton balls, and the Transwell bottom chamber was observed under an inverted microscope (IX71, Olympus). Transwell migration experiments did not require Matrigel coating, and the incubation period was 16 h. Cells from at least four randomly-selected visual fields were counted.
3
Tube Formation Assay for HUVECs
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After Matrigel (EMD Millipore) was thawed on ice; 96-well plates coated with 50 μL Matrigel in each well were incubated at 37°C for 30-min to allow the Matrigel to polymerize. To examine the effect of Nef-M1 peptide on tumor cell-induced tube formation of HUVECs, a conditioned medium was collected from Nef-M1 or sNef-M1 peptide treated HT29, MDA-MB231 cells and also from MDA-MB468 cells as indicated and used as the growth medium for HUVECs. A total of 1 × 104 HUVECs were seeded into each well that had conditioned medium. Cells were then incubated for 8 h to allow formation of tube-like structures. Endothelial cell tube formation was assessed with an inverted photomicroscope. Tubular structures were quantified by manually counting the number of capillaries in low-power fields.
4
3D Matrigel Sandwich Culture of Neuroblastoma Cells
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3D multicellular spheroids were cultured based on a previous study [9 (link)]. A modified protocol was developed to form the Matrigel sandwich structure for 3D in vitro models, based on the previous reports from our lab [11 (link)] as shown in Fig. 1 . Briefly, to form the bottom layer of Matrigel, 50 µL Matrigel (Sigma-Aldrich) was added into each well which had been pre-chilled on ice, and then the plates were incubated at 37 °C for 30 min, allowing the Matrigel to polymerise. SH-SY5Y cells in 10% Matrigel (v/v) in 500 µL pre-chilled EBM-2 supplemented with 2% FBS and 1% penicillin–streptomycin were added onto the polymerised Matrigel at the density of 3–5 × 104 cells/mL. In the co-culture model, HUVECs were added together with SH-SY5Y onto the polymerised gel layer at the density of 1–1.5 × 105 cells/mL in 500 µL endothelial basic medium (EBM-2, Lonza, UK) supplemented with 2% FBS (Life Technologies, US) and 1% penicillin–streptomycin (Thermo Fisher). The 3D in vitro systems were maintained at 37 °C, 5% CO2 for at least 24 h before further drug testing.![]()
Schematic procedures for 2D and 3D in vitro models used in this study.
5
Sciatic Nerve Crush Injury and MMP Modulation
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A total number of 18 SD rats were divided into three groups and exposed to sciatic nerve crush injury as previously mentioned. Rats in each group received the injection of 10 μl of a 1:1 volume mixture of Matrigel (BD Biosciences, Billerica, MA, United States) with 100 nM human recombinant MMP7 protein (Merck Millipore, Darmstadt, Germany), Matrigel with 100 nM human recombinant MMP9 protein, or Matrigel with 0.9% sodium chloride, respectively, at the crush site immediately after sciatic nerve crush. Rats were sacrificed by decapitation at 4 days after treatment. Sciatic nerve segments (3-mm-long crushed part) were collected for subsequent quantitative real-time PCR to determine the mRNA abundances of CLDN1, CLDN10, and CLDN22.
6
Matrigel-Based Cell Migration Assay
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Matrigel was diluted to 1 mg/ml with precooled serum-free medium; 100 µl Matrigel (EMD Millipore) was added to the lower chamber of the Transwell inserts, and then incubated at 37°C for 1 h. After culturing for 24 h in serum-free medium, the cells were collected and added to the upper chamber, and medium containing 20% FBS was added to the lower chamber. After 24 h, the inserts were washed twice with PBS and fixed with 100% methanol for 10 min at room temperature. Then, the cells in the upper chamber were removed with cotton swabs, and the remaining cells were stained with 0.1% crystal violet solution for 20 min at room temperature. The cells were counted under a light microscope (magnification, ×50). At least 5 random fields were observed, from which the average value was recorded.
7
Wound Healing and Cell Invasion Assays
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Cells were seeded at 4×105 cells/well in 6-well plates and incubated for 24 h at 37°C. A wound was made by scratching a straight line in the monolayer of cells and NEDD4-siRNA transfection was carried out. At 0, 24 and 48 h, dead cells were removed by washing with PBS and the migration ability was observed using an Olympus BX61 upright microscope equipped with imaging technology (Olympus Corporation, Tokyo, Japan). Migration distance was measured and migration area was calculated automatically by using the Image J version 1.42 software (National Institutes of Health, Bethesda, MA, USA).
Cell invasion assays were carried out using the Transwell chamber assay with Matrigel (EMD Millipore, Billerica, MA, USA) (14 (link)). Matrigel (Sigma-Aldrich; Merck KGaA) was added to the filter to form a thin gel layer. Following incubation at 37°C for 48 h, the cells adherent to the upper surface of the filter were removed using a cotton applicator, then stained with crystal violet. Ten random fields were selected and the values obtained were calculated by averaging the total numbers of cells from triplicate determinations.
Cell invasion assays were carried out using the Transwell chamber assay with Matrigel (EMD Millipore, Billerica, MA, USA) (14 (link)). Matrigel (Sigma-Aldrich; Merck KGaA) was added to the filter to form a thin gel layer. Following incubation at 37°C for 48 h, the cells adherent to the upper surface of the filter were removed using a cotton applicator, then stained with crystal violet. Ten random fields were selected and the values obtained were calculated by averaging the total numbers of cells from triplicate determinations.
8
Evaluating Cell Colonization on Coated PCL Fibers
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To investigate whether prior coating of PCL fibers will optimize colonization, 5.3 × 103 cells/cm2 ECFCs and 2.6 × 104 cells/cm2 pMSCs were seeded on coated PCL 24-well plates. For ECFCs, plates were coated with either Matrigel (1:120 in PBS) or collagen I (1:71.5 in acetic acid); for pMSCs, plates were coated with either Matrigel or gelatin (0.1% gelatin (Merck, Darmstadt, Germany). Uncoated plates were used for each cell type as reference. Seeding of PCL plates was performed as described before [55 (link)]. On days 1, 4, 7, and 14 for ECFCs and on days 1, 4, 7, 14, 21 for pMSCs, plates were analyzed using a NyOne image cytometer (Synentec, Elmshorn, Germany) and scanning electron microscopy.
9
HUVEC Tube Formation and Invasion
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For tube formation, 2 × 104 HUVEC in 100 µL of conditional medium (cell culture supernatant) were seeded into a 96‐well containing 50 µL Matrigel (Merck Millipore) and incubated at 37°C with 5% CO2 for 4 h. The tube photos were taken by inverted microscope and branch points in each frame were counted. For invasion assay, 50 µL Matrigel (Merck Millipore) diluted with DMEM (1 : 5 dilution) was added into a Transwell insert (8.0 µm; Corning, Kennebunk, ME, USA) and 2 × 104 HUVEC in 100 µL of conditional medium (cell culture supernatant) was seeded into the coated Transwell. After 24 h of incubation, non‐invaded cells were removed with a cotton swab from the upper part of the Transwell and inserts were fixed with 4% paraformaldehyde for 15 min at room temperature. The inserts were stained in 500 µL of 0.03% crystal violet solution (Beyotime) and sheeted with neutral balsam (Beyotime). Five pictures were taken for each insert and the number of invaded cells in each frame was counted.
10
Matrigel-Based Tube Formation Assay
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Matrigel (ECM625, Merck Millipore, USA) was thawed on ice overnight, dispensed onto 96-well plates (50 μL/well) and polymerized at 37°C for 1 h. HUVECs (2×104 cells/well) were seeded onto the Matrigel layer and cultured in HUVEC culture medium or tumor cell-conditioned medium that was collected earlier at a 1:1 ratio. Tube formation was observed after 7 hours’ incubation at 37°C, and imaging was performed by an inverted microscope (Olympus Corporation, Tokyo, Japan). The results, including the number of tube nodes (the intersection among 3 or more tubes) in a 100× field, were analyzed by using ImageJ software.
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