Liquid matrigel (50 μL, BD, cat.: 354,230) was added into pre-cooled 96-well plates, and then incubated at 37°C for 45 min. HRCEC received hypoxia or/and PF treatments were seeded on the matrigel (5 × 104 cells/well). After 4 h of incubation at 37°C, the growing blood vessels were visualized under microscope (Olympus; BX53M) [21 (link)].
Liquid matrigel
Manufactured by BD
Sourced in United States
Liquid Matrigel is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma. It is a complex mixture of extracellular matrix proteins, growth factors, and other components that support the growth and differentiation of various cell types. Liquid Matrigel is commonly used as a substrate for cell culture and tissue engineering applications.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel matrix cat, by BD (3 mentions)
Phase contrast microscope, by Leica (2 mentions)
Bx53m, by Olympus (1 mentions)
Matrigel, by BD (1 mentions)
Eclipse ts100, by Nikon (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dnase type 1, by Merck Group (1 mentions)
Nod scid mice, by Charles River Laboratories (1 mentions)
Fitc uea 1, by Merck Group (1 mentions)
Dii ac ldl, by Biomedical Technologies (1 mentions)
8 protocols using liquid matrigel
1
In Vitro Angiogenesis Assay
2
In Vitro Tube Formation Assay
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Liquid Matrigel (BD Biosciences, USA, BD Matrigel Matrix Cat. No. 356234) was added into 96-well tissue culture plates after the induction of MSCs differentiation into blood vascular cells. The in vitro tube formation assay was performed using the Matrigel (BD Bioscience, USA) according to the manufacturer's instructions. Cells were seeded into the Matrigel-coated 96-well plate, and after required treatments for 72 h of hypoxic culture, images were captured using a bright-field microscope. The observed tubes were counted [25 (link)]. Four representative fields are counted and the average of the total area of complete tubes formed by cells per unit area is compared by Image-Pro Plus®.
3
Modeling Angiogenesis using HUVECs
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When cultured on a tree-dimentional membrane-basement matrix, HUVECs are able to form capillary like structures, mimicking the events occurring during vessel lumen formation in vivo. A 24-microwell plate, pre-chilled at −20°C, was carefully filled with 300 μl/well of liquid matrigel (10 mg/ml, BD Biosciences, Milano) at 4°C with a pre-chilled pipette. The matrigel was then polymerized for 1 h at 37°C. 5 × 104 HUVEC, pre-treated with XN or EGCG (range 5 to 20 μM) for 24h, were suspended in 1 ml of complete medium and layered on the top of the polymerized matrigel. For the experiments using the STO-609 compound (20 μM), cells were pre-treated for 30 minutes, then treated with 10 μM XN for 1 hour. After 6 h of incubation at 37°C, the formation of capillary-like networks was examined under an inverted microscope (Nikon Eclipse TS100), equipped with charge-coupled device optics and a digital analysis system. The length of segments were quantified by the ImageJ software, using the “Angiogenesis Analyzer” tool [64 ].
4
Primary Cytotrophoblast Differentiation into Extravillous Trophoblasts
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Primary CTBs were isolated as previously reported.17 For the in vitro differentiation of primary CTBs into EVTs, 2.5 × 106 CTBs were placed into 35‐mm dishes precoated with 1 mL of liquid Matrigel (BD Biosciences) at 1 mg/mL and maintained in DMEM/F12 (Gibco) containing 10% foetal bovine serum (FBS, Gibco) for 48 hours.
5
Isolation and Differentiation of Cytotrophoblasts
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The placental villi (5-7 weeks of gestation, 20 placentas each time, n=5) were minced and digested with 0.25% trypsin and 0.02% DNase type I (Sigma-Aldrich) four times at 37°C to detach the CTBs. The cell suspension from the first digestion was discarded, and those from the following three digestions were carefully loaded onto a Percoll gradient (70% to 10%, in 10% steps), then centrifuged at 1200 g for 25 min. Cells that sedimented at the 30%-40% region of the Percoll gradient were collected (94.6% were CTBs). To differentiate CTBs into EVTs, CTBs were seeded onto a six-well plate coated with liquid matrigel (1 μg/μl; BD Biosciences) at 2.5×106 cells per well and cultured in DMEM/F12 supplemented with 10% fetal bovine serum for 48 or 72 h. After 48 h of culture, 96% of the cells were induced EVTs.
6
Tumor Growth Inhibition by Anti-VEGF mAb
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Procedures involving animals and their care were conformed to institutional guidelines that comply with national and international laws and policies (EEC Council Directive 86/609, OJ L 358, 12 December, 1987) and were authorized by the Italian Ministry of Health (authorization no. 617/2016-PR). For tumor establishment, eight-week-old nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice (Charles River; Wilmington, Massachusetts, USA) were subcutaneously (s.c.) injected into both flanks with 0.3−0.5 × 106 tumor cells mixed at 4 °C with liquid Matrigel (BD; Franklin Lakes, NJ, USA). Tumor volume (mm3) was calculated as previously reported [20 (link)]. When tumors were about 150 mm3, anti-human VEGF mAb (bevacizumab) was administered i.p. at 100 µg/dose bi-or tri-weekly to NOD/SCID and mice were sacrificed 48 h after the last treatment. Control mice received i.p. injections of PBS.
7
In Vitro Angiogenesis Assay
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Liquid Matrigel (BD Biosciences, USA, BD Matrigel Matrix Cat. No. 356234) was added into 96-well tissue culture plates after the induction of EC differentiation into blood vascular cells. ECs (2 × 105 cells per well) were grown in a final volume of 0.30 mL culture medium containing 150 mL M199 (GibcoBRL) and 150 mL CM. Each well was digitally photographed under a phase contrast microscope (Leica). The observed tubes and branching points were counted [44 (link)]. Four representative fields are counted and the average of the total area of complete tubes formed by cells per unit area is compared by Image-Pro Plus®.
8
Quantifying Angiogenic Potential of Cells
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Liquid Matrigel (BD Biosciences, USA, BD Matrigel Matrix Cat. No. 356234) was added into 96-well tissue culture plates after the induction of EC differentiation into blood vascular cells. Each well was digitally photographed under a phase contrast microscope (Leica) . The observed tubes and branching points were counted [41] (link).
Acetylated low-density lipoprotein (acLDL) uptake and Ulex europaeus agglutinin-1 (UEA-1) binding test After the induction of MSC differentiation into blood vascular cells, the cells were characterized as adherent cells that were double-positive for DiI-acLDL (Biomedical Technologies, USA) uptake and uorescein isothiocyanate (FITC)-UEA-1 (Sigma) binding. Cell nuclei were counterstained using DAPI [48] .
Acetylated low-density lipoprotein (acLDL) uptake and Ulex europaeus agglutinin-1 (UEA-1) binding test After the induction of MSC differentiation into blood vascular cells, the cells were characterized as adherent cells that were double-positive for DiI-acLDL (Biomedical Technologies, USA) uptake and uorescein isothiocyanate (FITC)-UEA-1 (Sigma) binding. Cell nuclei were counterstained using DAPI [48] .
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