Human umbilical vein endothelial cells (HUVECs) were cultured in a-MEM containing 10% FBS in a humidified, 5% CO2 incubator at 37°C. For the cell migration assay, HUVECs were starved overnight with a serum-free culture medium, and then 2 × 105 cells/well were seeded into a six-well plate for attachment. Afterwards, pipette tips were used to make an artificial scratch at the center of the cellular monolayer. The migration of cells was photographed at 0 and 24 h after the cell culture medium was replaced with various extracts. The migration rate (%) was calculated as follows: migration ratio (%) = [(original distance—remaining distance)/original distance] × 100%. For the transwell migration assay, 2 × 104 cells/well were seeded in the upper chamber with 8 μm pore filters (Corning), and different sample extracts were added to the lower chamber as the chemoattractant for 24 h. The cells that migrated to the opposite side of the upper chamber were fixed and stained with 0.2% crystal violet, and the staining cells were imaged for analysis. For the RT-PCR assay, total RNA was isolated from the adherent HUVECs treated with different sample extracts. The mRNA expression levels of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor a (HIF-α) (angiogenesis-related genes) were analyzed using RT-PCR.
8 μm pore filter
Manufactured by Corning
Sourced in United States
The 8 μm pore filters are laboratory equipment designed to filter and separate particles or components from a liquid or gas sample. These filters feature a pore size of 8 micrometers, which allows for the effective removal of larger particles while permitting the passage of smaller components. The filters are built to provide reliable and consistent filtration performance across a variety of laboratory applications.
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Lab products found in correlation
Matrigel, by BD (5 mentions)
Crystal violet, by Beyotime (2 mentions)
Matrigel basement membrane matrix, by BD (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Bgj398, by Santa Cruz Biotechnology (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Basic fibroblast growth factor (bfgf), by Merck Group (1 mentions)
Transwell insert, by Corning (1 mentions)
Inverted microscope, by Nikon (1 mentions)
Cxcl10, by R&D Systems (1 mentions)
Gelatin, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
24 well transwell chamber, by Corning (1 mentions)
Boyden chamber, by Corning (1 mentions)
15 protocols using 8 μm pore filter
1
Evaluating Angiogenic Potential of Extracts
2
Transwell Migration and Invasion Assay
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Transwell migration and invasion assays were carried out with 12-well transwell plates containing 8-μm pore filters (Corning, New York, NY, USA). For invasion assays, the bottom of transwell chamber was coated with BD Matrigel Basement Membrane Matrix (BD Biosciences, San Diego, CA, USA). 1× 105 cells were seeded into the upper chamber in 500 μl serum-free basic medium, and the lower chamber was filled with 1500 μl complete culture medium. The migration and invasion systems were incubated for 24 h and 48 h at 37 °C respectively. Cells on the upper side of the chambers were removed by scrubbing, and cells on the lower surface of the membrane were fixed with 4% paraformaldehyde for 15 minutes. Then it was stained with 0.1% crystal violet (Beyotime Institute of Biotechnology, Jiangsu, China) for 30 minutes. The infiltrating cells were viewed and counted in five randomly selected fields under a microscope.
3
Transwell Assay for Cell Migration and Invasion
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Cell migration was assayed using the transwell method, with 8-μm pore filters (Corning, NY). The lower chamber was filled with Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS), and 2 × 104 cells in 0.5 ml of DMEM were loaded into the upper chamber. After a 22-hour incubation period, the cells that migrated to the bottom of the membrane were fixed with 4% formaldehyde. The cells on the top of the membrane were removed by wiping the surface with a cotton swab. The cells were stained with 0.5% crystal violet and observed under a microscope. The number of migrated cells was counted at a magnification of 200× from five adjacent microscope fields. For the Matrigel invasion assay, the procedures used were the same as those described above, except that the transwell membrane was coated with Matrigel (BD, CA, USA) to form a matrix barrier.
4
Boyden Chamber Cell Migration Assay
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Cells (8×104) in 500 μL DMEM containing 1% FBS were seeded into the upper part of the Boyden chamber with 8-μm pore filters (Corning, Cambridge, MA). Cell migration was induced by 10% FBS (GIBCO) or 25 ng/ml bFGF (Sigma) in the lower part. In some experiments, 1 μM BGJ398 (Santa Cruz Biotechnology) or 0.1% DMSO was included in the upper-chamber medium. After 48 h, cells that had migrated to the lower surface of the membrane were fixed and stained with 0.5% crystal violet (Sigma) and counted under a microscope in six random fields.
5
Cell Migration and Invasion Assay
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For cell migration assay, transfected Hec-1B cells (5× 104) were suspended in 200 μL of serum-free medium and seeded onto the upper chambers of the 24-well plates with 8-μm-pore filters (Corning, NY, USA). 20% fetal bovine serum was added to the lower chamber medium. For cell invasion assay, the membranes of the top chambers were coated with Matrigel (BD, USA). After using drugs for 18-24h, the crossed cells were fixed, stained and counted at 100×magnifcation.
6
Cell Migration Assays for BMSCs and HUVECs
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To evaluate cell migration, wound healing assay and Transwell assay were conducted. For wound healing assay, BMSCs or HUVECs were seeded in 24-well plates at a density of 5000 cells/well. Twenty-four hours after cells being seeded, a series of 1.5 mm-linear wound were scratched by blue tip and washed with PBS for three times. Culture medium containing 10 μg/mL exosomes (Exo or H-Exo) and 1% FBS were added to continue the incubation for another 24 h. Wound closer was observed by an optical microscope and analyzed using ImageJ software. For transwell assay, 1 × 105 BMSCs or HUVECs were seeded onto the upper chamber with 8-μm pore filters (Corning). Conditional medium (500 μL) containing 10 μg/mL exosomes (Exo or H-Exo) was added. After incubated for 24 h, cells were fixed with 4% paraformaldehyde and unmigrated cells were gently wiped using a cotton tip. Then, 1% crystal violet was used to stain the migrated cells for 30 min. The relative numbers of migrated cells were counted and analyzed in five randomly microscopic fields per filter.
7
Transwell Invasion Assay for TNBC
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The transfected TNBC cells were harvested and put into the top chamber of transwell inserts with 8 μm pore filters (Corning, Corning, NY, USA) and Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) in 24-well plates. After 48 h, the fixed invasive cells were treated with crystal violet staining and counted by microscope at 200× magnification.
8
Modified Boyden Chamber Assay for MSC Migration
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Three types of MSCs were examined by modified Boyden chamber assay using transwell plates with 8 μm pore filters (Corning, USA). The cells were seeded into the upper chambers, after which gliomas-CM was added to the lower chambers. Following incubation for 6 h, cells that had not migrated through the upper side of the filters were scraped off with swabs. The filters were then stained with hematoxylin, after which cells that had migrated to the lower side were counted using an inverted microscope (Nikon Co., Japan). The migration assay was conducted in triplicate.
9
Immunosuppressant Effects on MSC Migration
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Effect of immunosuppressants on MSC migration was examined in transwell plates with 8 μm pore filters (Corning, Cambridge, MA, USA) [14 (link)]. Upper wells were precoated with 0.1% gelatin (Sigma-Aldrich) for 2 h at 37°C. Upper wells were loaded with 2 × 104 MSCs in 200 μl of medium and the lower wells with 500 μl of medium containing 10% fetal bovine serum and 100 ng/ml CXCL10 (R&D Systems). After 24 h, nonmigrated cells on the upper side of the filters were removed and the membranes were fixed in 10% formalin. MSCs that migrated to the lower side of the filter were stained with 0.5% crystal violet for 10 min and were counted under an inverted light microscope.
10
Cellular Proliferation and Migration Assays
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To assess the proliferation ability of cells, CCK8 analysis was conducted. Specifically, every well introduced 1000 cells. And the blank group served as a control without any cells. The mixture of CCK8 solution and culture medium was added to the wells of cells and incubated at a temperature of 37 °C with 2 h. To calculate cell survival/proliferation, we subtracted the optical density (OD) values of the blank wells from that of the test wells using microplate readers.
In order to conduct scratch wound assays, a total of 2 × 105 cells were plated until confluence was achieved. The detachment of cells from the monolayer was addressed by scratching it using a tip and subsequently washing it. To assess tumor cell migration, all cells were photographed 24 or 36 h after the wounding process depending on the degree of cell growth.
For the transwell invasion assay, the base of the chamber with 8 μm pore filters (Corning) is preloaded with matrigel. Then, a suspension of 2 × 104 cells per well was prepared in a 2% FBS medium and then seeded into the upper chamber. After 48 h, the upper chambers were removed from a 10% FBS medium from the bottom 24-well and washed, with the cells fixed and stained.
In order to conduct scratch wound assays, a total of 2 × 105 cells were plated until confluence was achieved. The detachment of cells from the monolayer was addressed by scratching it using a tip and subsequently washing it. To assess tumor cell migration, all cells were photographed 24 or 36 h after the wounding process depending on the degree of cell growth.
For the transwell invasion assay, the base of the chamber with 8 μm pore filters (Corning) is preloaded with matrigel. Then, a suspension of 2 × 104 cells per well was prepared in a 2% FBS medium and then seeded into the upper chamber. After 48 h, the upper chambers were removed from a 10% FBS medium from the bottom 24-well and washed, with the cells fixed and stained.
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