Tube formation assays were performed using HUVEC cells as described elsewhere [34 (link)]. Briefly, for tube formation assays, HUVECS were transfected with Jarid2 constructs (0.25μg) using a nucleofector kit (Lonza), followed by plating 0.6 × 105 cells on a 24-well plate coated with Low Growth Factor Matrigel (BD Biosciences, San Jose, CA) and supplemented with 50ng/ml of VEGF165 (R&D Systems, MN) in serum-free media. The tubes were imaged at 10X magnification and quantified from 4 different fields. Co-transfection of Jarid2 and miR-130a (100nM) was performed as described above.
Low growth factor matrigel
Manufactured by BD
Low Growth Factor Matrigel is a gelatinous protein mixture that is derived from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma. It is a reconstituted basement membrane preparation that lacks key growth factors typically found in Matrigel, making it suitable for applications where reduced growth factor signaling is desired.
Automatically generated - may contain errors
Lab products found in correlation
Minimal essential amino acids, by Thermo Fisher Scientific (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
Nucleofector kit, by Lonza (1 mentions)
Vegf165, by R&D Systems (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
B 27 supplement without vitamin a, by Thermo Fisher Scientific (1 mentions)
Y 27632, by Merck Group (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Noggin, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by R&D Systems (1 mentions)
R spondin, by R&D Systems (1 mentions)
4 protocols using low growth factor matrigel
1
HUVEC Tube Formation Assay with Jarid2 and miR-130a
2
Sildenafil Modulates HUVEC Angiogenesis
3
Sildenafil Modulates HUVEC Angiogenesis
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Human umbilical vein endothelial cells, HUVEC, were grown in Dulbecco’s Modified Eagles Medium (DMEM) that was supplemented with 5% FBS, antibiotic-antimycotic (Invitrogen) and minimal essential amino acids (Invitrogen). Capillary tube formation was conducted in 6 well culture plates coated with low growth factor matrigel (BD Biosciences). HUVEC cells were seeded at 1×104 cells/cm2 in DMEM with 5% FBS with, or without, sildenafil (100 nM) and cells were imaged at various times. Capillary tube formation was quantified in 3 random areas of the well and the length of cords between cells was measured using ImageJ Angiogenesis Analyzer.
4
Jejunal Crypt Isolation and Culture
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Jejunal crypts were isolated and in vitro crypt culture was performed as previously described [32 (link)]. Approximately 30 crypts were plated into 24 well plates in 10μl Low Growth Factor Matrigel (BD Biosciences, Franklin Lakes, NJ) containing 50ng/ml EGF (R&D Systems, Minneapolis, MN), 100ng/ml Noggin (Peprotech, Rocky Hill, NJ) 1μg/ml R-spondin (R&D Systems, Minneapolis, MN) and 10uM Y-27632 (Sigma-Aldrich, St. Louis, MO). After polymerization 250μl Advanced DMEM-F12 containing N2 supplement (Invitrogen, Carlsbad, CA), B27 supplement without vitamin A (Invitrogen, Carlsbad, CA), 10mM HEPES (Invitrogen, Carlsbad, CA), Glutamax (Gibco-Invitrogen, Carlsbad, CA) and Pen/Strep (Gibco-Invitrogen, Carlsbad, CA) was added to each well. Growth factors were added every 2 days at the same concentrations as the initial plating except R-spondin, which was reduced to 500ng/ml. Medium was changed every 4 days. The number of enterospheres or enteroids and their complexity was recorded at days 1, 4 and 8-post plating. Representative images of cultures were taken using an inverted fluorescent microscope (Olympus IX83).
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