Human umbilical vein endothelial cell migration and invasion were analyzed using transwell inserts (8-μm pore size; Corning) according to the manufacturer’s protocol. In brief, HUVECs were plated in the upper chamber, which was uncoated (1 × 105 cells in 200 μl serum-free DMEM for the migration assay) or coated with 100 μl of Matrigel (BD Matrigel 356234) diluted 8× in DMEM (2 × 105 cells in 200 μl serum-free DMEM for the invasion assay), and DMEM containing 10% FBS (500 μl) was added to the lower chamber as a chemoattractant. Subsequently, the HUVECs were treated with HCC-Exos or 1× PBS. After 24 h of incubation at 37°C and 5% CO2, the cells on the upper side of the membrane were removed with cotton swabs. Following this, the migrated or invaded cells on the underside of the membrane were fixed in 4% paraformaldehyde for 20 min at room temperature and then stained with 0.1% crystal violet for 10 min. Five random fields were imaged and counted. The experiments were performed in triplicate independently.
Matrigel 356234
Manufactured by BD
Sourced in United States
BD Matrigel 356234 is a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. It is used as a substrate for the in vitro culture of various cell types, such as epithelial cells, endothelial cells, and stem cells.
Automatically generated - may contain errors
Lab products found in correlation
Transwell insert, by Corning (1 mentions)
Inverted phase contrast microscope, by Olympus (1 mentions)
Dulbecco modified eagle medium (dmem), by Corning (1 mentions)
Rpmi 1640, by Corning (1 mentions)
C57bl 6n, by Charles River Laboratories (1 mentions)
Flag m2 3165, by Merck Group (1 mentions)
Cd9 sc13118, by Santa Cruz Biotechnology (1 mentions)
E cadherin 20874 1 ap, by Proteintech (1 mentions)
4 protocols using matrigel 356234
1
Evaluating HUVEC Migration and Invasion
2
Angiogenic Potential of Samples
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Tube formation assay, a classic angiogenesis assay based on the angiogenic differentiation of endothelial cells, was selected in our study to exam the angiogenic effect on HUVECs of each sample. One day before experiment, pipets, 24-well culture plates and sterile tips were cooled in −20 °C. Then thaw the Matrigel (BD Matrigel™356234, USA) at 4 °C, keep all the procedure on ice. Coated 24-well plates with 300 μL per well of Matrigel and warmed in 37 °C incubator for 15 min to allow gelling of the Matrigel. Added 4 × 106 HUVECs within 1 mL of sample extracts into each cell, further cultured for 4 h. Then observe the tube structures under a phase contrast inverted microscope (Olympus, Germany). Total branch length was analyzed by image J software with Angiogenesis Analyzer plug.
3
Tube Formation Assay with HUVECs
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HUVECs were harvested after 24 h cocultured, and cells were (3 × 104 cells per well) seeded onto matrigel plates (Matrigel356234; BD Biosciences) and cultured at 37 °C in 5% CO2. And observed by an inverted microscopy over the course of 24 h for tube formation. Networks formed by the HUVECs were quantified with Image J software (National Institutes of Health, Bethesda, MD, USA).
4
Cell Line Authentication and Characterization
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SW480, SW620, HCT116, DLD1, LOVO, HT29, HEK293, and B16 cell lines were obtained from the ATCC and recently been authenticated by STR profiling. The SW480 cell line was cultured in RPMI 1640 (Corning) supplemented with 10% FBS (PAN, P30-3302), and the SW620, HCT116, DLD1, LOVO, HT29, HEK293 as well as B16 cell lines were cultured in DMEM (Corning) supplemented with 10% FBS. These cell lines were cultured in a 37 °C incubator with 5% (v/v) CO2.
Mouse monoclonal anti-SEC23B antibody was generated against the synthetic peptide LTKPAMPMQQARPAQPQEHP, and was validated in our laboratory (Supplementary Fig.1a ). The commercial antibodies used in this study included GFP (RM1008), GAPDH (RM2002), and α-Tubulin (RM2007) antibodies from Sungene Biotech; EPCAM (66316-1-AP), E-cadherin (20874-1-AP) and PDI (11245-1-AP) from Proteintech; FLAG (M2-3165) from Sigma-Aldrich; GM130 (A5344) from ABclonal; CD9 (sc13118) from Santa Cruz.
The reagents used in this paper included Fibronectin (354008) from Biocoat, Matrigel (356234) from BD, puromycin (sc-205821A) from Santa Cruz, MG132 (Carbobenzoxy-L-leucyl- L-leucyl-L-leucinal) (MB5137) from EPSILON, and chlorhexidine (CHX) (C7698) from SBJBIO, anti-fade mounting reagent (C1210) from Polygen.
C57BL/6N was purchased from Charles River Laboratories.
Mouse monoclonal anti-SEC23B antibody was generated against the synthetic peptide LTKPAMPMQQARPAQPQEHP, and was validated in our laboratory (Supplementary Fig.
The reagents used in this paper included Fibronectin (354008) from Biocoat, Matrigel (356234) from BD, puromycin (sc-205821A) from Santa Cruz, MG132 (Carbobenzoxy-L-leucyl- L-leucyl-L-leucinal) (MB5137) from EPSILON, and chlorhexidine (CHX) (C7698) from SBJBIO, anti-fade mounting reagent (C1210) from Polygen.
C57BL/6N was purchased from Charles River Laboratories.
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