The 96-well plates were coated with pre-cooled matrigel (80 μL/well; BD Biosciences, Bedford, MA, USA) and incubated for 30 min at 37°C. HCMEC/D3 cells (5 × 104) were added to the plate and cultured with transfected U251 and A172 cells’ medium for 24 h. After co-culture, the tube formation was photographed under a microscope, and angiogenesis was quantitated by counting the tubule length.
Precooled matrigel
Manufactured by BD
Sourced in United States
Precooled Matrigel is a solubilized basement membrane matrix preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma. It is provided in a pre-cooled state to maintain its liquid form and ease of handling. Matrigel is commonly used as a substrate for culturing cells in vitro.
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Lab products found in correlation
8 protocols using precooled matrigel
1
Angiogenic Co-culture Assay
2
In vivo Tumorigenicity Assessment of GSCs
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A total of 12 male BALB/c nude mice (5-weeks-old) were obtained from Beijing HFK Bioscience Co., Ltd. (Beijing, China) and kept in a specific pathogen-free vivarium with temperature between 26–34°C, humidity at 30–50%, 12/12 h light/dark cycle and free access to food and water (n=6/group). For the in vivo tumorigenicity assessment of the GSC-vector and GSC-miR-141 cell lines, ~1.0×106 GSCs were cultured, rinsed with precooled PBS three times, and resuspended with precooled Matrigel (BD Biosciences, San Jose, CA, USA) diluted with PBS at a ratio of 1:3. This was subsequently injected into either the right or left axillary fossa of each mouse. From day 7, mouse tumor volumes were measured with calipers by two experienced lab members at different time points throughout the day, every 3 days. The formula to calculate tumor volume was volume=0.5ab2, where ‘a’ is the long axis and ‘b’ is the short axis of the tumor. All animals were sacrificed using general anesthesia by 1.5% isoflurane (Sigma-Aldrich; Merck Millipore) inhalation and tumors (maximum size, 4.7 mm3) were weighed.
3
In Vitro Tube Formation Assay
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Pre-cooled Matrigel (BD Biosciences, USA) was loaded onto the 24-well plate for solidification at 37°C in 30 min. Then, HUVECs were digested, counted, and adjusted to the cell density of 4×105 cells/ml. Next, 100 μl of cell suspension was put into the matrigel-coated 96-well plate and cells were cultured at 37°C for 24 hr in a CO2 incubator. The formation of tubes was observed under a phase contrast microscope (Olympus) and photographed randomly under 100 fold microscope. The tube lengths were analyzed using Image-Pro Plus software, version 5.0 (BD Biosciences).
4
HUVEC-RFP Tube Formation Assay
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Red fluorescent proteins (RFP) expressing human umbilical vein endothelial cells (HUVECs) were obtained from Angio-Proteomie (Boston, USA) and cultured in endothelial growth medium (EGM-2) and passage 5 was used for the experiments. 50 μl of precooled Matrigel (BD, Corning, USA) was coated into each well of a 96-well plate and polymerized for 30 min at 37 °C. Next, 2 × 104 HUVEC-RFP cells were suspended in a mixture of conditioned medium (50 μl) and endothelial cell medium (50 μl) containing 10% FBS. The fluorescence detection of tube formation was photographed using inverted microscopy (IX-53, Olympus) after 6 h of incubation at 37 °C.
5
Angiogenesis Assay with VEGF Microparticles
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Approximately 96 well plates were coated with 50 µL/well of precooled Matrigel (BD Biosciences) and incubated in a humidified incubator (37 °C, 5% CO2) for 30 min for the Matrigel to solidify. Then, 1 × 104 cells/well were seeded with and without rVEGF121 (200 and 500 ng), 100µL of VEGF-loaded PLA microparticles (day 1, 20, and 30), and PBS-loaded PLA microparticles (negative control) and incubated for 4 h. After the incubation, CalceinAM (Trevigen), a fluorescent monitoring tube formation and a cell-permeable dye were added at a final concentration of 2 µg/mL and incubated for 30 min at 37 °C and provided with 5% CO2 in a light-free environment. The cell conditions and tube formation were monitored under a Nikon Laser Scanning Confocal microscope model C2 at 4× magnification, and the image was captured in the camera provided with the microscope. The number of tubes formed were analyzed using WimCam online software (Wimasis, Onimagin Technologies SCA, Spain).
6
Angiogenesis Assay on Matrigel
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The 48-well plate
was coated with 150 μL of precooled matrigel (BD Sciences, 354234)
on ice and then placed at 37 °C for 30 min to allow the gelling
of matrigel. Then collect HA-Dll4 expression HUVECs or primary HUVECs
and seed them at a density of 10 × 104 cells/well
and 5 × 104 cells/well in cell culture media with
the required concentration of BPEI 25K, respectively. After incubation
at 37 °C, 5% CO2 for 6 to 24 h, the tube formation
of cells on matrigel was imaged with an Olympus IX81 live cell imaging
system equipped with a 10× objective. The five random fields
of tubule-like structures in images were quantified by the angiogenesis
plugin of ImageJ software.
was coated with 150 μL of precooled matrigel (BD Sciences, 354234)
on ice and then placed at 37 °C for 30 min to allow the gelling
of matrigel. Then collect HA-Dll4 expression HUVECs or primary HUVECs
and seed them at a density of 10 × 104 cells/well
and 5 × 104 cells/well in cell culture media with
the required concentration of BPEI 25K, respectively. After incubation
at 37 °C, 5% CO2 for 6 to 24 h, the tube formation
of cells on matrigel was imaged with an Olympus IX81 live cell imaging
system equipped with a 10× objective. The five random fields
of tubule-like structures in images were quantified by the angiogenesis
plugin of ImageJ software.
7
Tube Formation Assay with Conditioned Media
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Conditioned media from HNE1 and CNE2 cells were collected. Precooled Matrigel (BD Biosciences) was added to precooled 96-well plates (50 µL per well) and allowed to solidify at 37 °C for 30 min. HUVECs were seeded and incubated with conditioned media at 37 °C for 6 h. Tube formation was observed and captured using an inverted microscope (Olympus).
8
Angiogenic Potential of HUVEC-RFP
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Red uorescent proteins (RFP) expressing human umbilical vein endothelial cells (HUVECs) were obtained from Angio-Proteomie (Boston, USA) and cultured in endothelial growth medium (EGM-2) and passage 5 was used for the experiments. 50μl of precooled Matrigel (BD, Corning, USA) was coated into each well of a 96-well plate and polymerized for 30min at 37°C. Next, 2×10 4 HUVEC-RFP cells were suspended in a mixture of conditioned medium (50μl) and endothelial cell medium (50μl) containing 10% FBS. The uorescence detection of tube formation was photographed using a inverted microscopy (IX-53, Olympus) after 6h of incubation at 37°C.
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