Cell migration and invasion assay was performed using 8-μm pore size membranes (Corning Costar Corp, Cambridge, MA, USA). Cells (8 × 105 in 600 µl serum-free media) were placed into the upper well of the Transwell chamber for migration assays (without Martrigel, BD Biosciences, Franklin Lakes, NJ, USA) and invasion assays (with Martrigel). Next, 800 μl of media containing 10% FBS was added to the lower chamber as a chemoattractant. After a 36-h incubation, the cells that migrated or invaded to the lower compartment were fixed with methanol and stained with 0.5% crystal violet. Cells on the undersides of the filters were imaged with an inverted IX51 microscope (OLYMPUS, Japan) and counted using ImageJ software.
Martrigel
Manufactured by BD
Sourced in United States
Martrigel is a laboratory product used for the cultivation and analysis of cells. It provides a gelatinous, protein-rich substrate that supports the growth and maintenance of various cell types in vitro. The core function of Martrigel is to mimic the extracellular matrix found in living organisms, enabling researchers to study cellular behavior and interactions in a more physiologically relevant environment.
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Lab products found in correlation
9 protocols using martrigel
1
Transwell Assay for Cell Migration and Invasion
2
Transwell Invasion Assay Protocol
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The Transwell invasion assay was detected as previously described [16 (link)]. Briefly, the upper chambers (Corning, USA) were coated with diluted Martrigel (BD, USA). Afterwards, the SW620 and HT29 cells were seeded into the upper chambers with serum-free DMEM, then the Transwell chambers were incubated in wells filled with DMEM containing 10% FBS. After 24 h of incubation at 37 °C, the cells in the inner chambers and the remaining Matrigel were removed with cotton swabs, while the cells on the outside surface of the lower chambers were fixed using 4% PFA and stained with 0.1% crystal violet for 15 min. After washing twice with PBS, the cell numbers in the five randomly chosen fields were calculated under a microscope.
3
Transwell Assay for Cell Invasion and Migration
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Transwell assays were applied for cell invasion detection. Firstly, the apical chambers were pre-coated with Martrigel (BD Biosciences, Franklin Lakes, NJ, USA) under sterile condition for 30 min, and then each chamber was filled with 30 μL RPMI-1640 and placed in a CO2 incubator. HT29 and SW116 cells were detached, centrifuged, resuspended in serum-free medium, and diluted to cell suspension at 5 × 105cells/mL. Next, each basolateral chamber was filled with 500 μL 10% FBS-supplemented RPMI-1640, while each apical chamber was loaded with 200 μL cell suspension. Then the chambers were incubated in a 37 °C incubator with 5% CO2 for 48 h. Next, the chambers were taken out with the medium washed away by PBS, and the invaded cells were stained by crystal violet for 10 min and then had the superficial crystal violet rinsed away. The non-invaded cells in the apical chambers were wiped away using cotton swabs, and the invaded cells were photographed under the microscope and the cell number was calculated.
Migration of HT29 and SW116 cells was performed via Transwell assay as well and all the procedures were conducted as above stated but without pre-coating Matrigel in the apical chambers. The chambers were taken out for staining after 24 h of incubation.
Migration of HT29 and SW116 cells was performed via Transwell assay as well and all the procedures were conducted as above stated but without pre-coating Matrigel in the apical chambers. The chambers were taken out for staining after 24 h of incubation.
4
Transwell-based Cell Invasion Assay
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Transwell upper chambers (Corning) coated with diluted Martrigel (BD Bioscience) and serum-free DMEM, were employed to seed MCF-7 cells, and later were treated with incubation in DMEM wells containing 10% FBS. After 24-hour incubation at 37°C, cells in inner chambers and remaining Matrigel were subjected to removal with cotton swabs, whereas cells on surface of lower chamber outsides were treated with fixation with 4% PFA to stain with 0.1% crystal violet for 15 minutes. After wash twice with PBS, the number of cells in five selected areas by random was counted under microscopes.
5
Evaluating Cell Invasion Capacity
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The invasiveness of cells was evaluated by the Martrigel (BD Bioscience)-coated Transwell (Corning Inc.). Cells (1 × 105) were seeded in the upper chamber in serum-free DMEM, and DMEM containing 20% FBS was added to the lower chamber. After incubation for 48 h, the cells on the upper surface of the membrane in the upper chamber were gently removed with a cotton swab. Then, the cells on the bottom surface of the membrane were fixed with 4% paraformaldehyde (PFA) for 15 min and stained with 0.1% crystal violet for 10 min. After washing with PBS 3 times, the invaded cells on the filter membrane were imaged, and cells in 5 random images were counted under the microscope to determine the number of invaded cells.
6
Transwell Invasion Assay for Cell Migration
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The Transwell invasion assay was detected as previously described [16] . Brie y, the upper chambers (Corning, USA) were coated with diluted Martrigel (BD, USA). Afterwards, the SW620 and HT29 cells were seeded into the upper chambers with serum-free DMEM, then the Transwell chambers were incubated in wells lled with DMEM containing 10% FBS. After 24 h of incubation at 37 °C, the cells in the inner chambers and the remaining Matrigel were removed with cotton swabs, while the cells on the outside surface of the lower chambers were xed using 4% PFA and stained with 0.1% crystal violet for 15 min. After washing twice with PBS, the cell numbers in the ve randomly chosen elds were calculated under a microscope.
7
Transwell Invasion Assay for Cell Migration
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Transwell invasion assay was detected as previously described [16] . Brie y, the upper chambers (Corning, USA) were coated with diluted Martrigel (BD, USA). Afterwards, SW620 and HT29 cells were seeded into the upper chambers with serum-free DMEM, and then the transwell chambers were incubated in wells lled with DMEM containing 10% FBS. Following 24 h incubation at 37 ˚C, cells inner the chambers and the remaining Matrigel were removed with cotton swabs and the cells on the outside surface of the lower chambers were xed using 4% PFA, stained with 0.1% crystal violet for 15 min. After washing twice with PBS, cell numbers in ve randomly chosen elds were calculated under the microscope.
Statistical analysis SPSS 22.0 statistical software was employed for the statistical analysis. Data was shown as mean ± the standard deviation (SD). The signi cant differences between groups were estimated by One-Way ANOVA. P < 0.05 was considered as statistically signi cant.
Statistical analysis SPSS 22.0 statistical software was employed for the statistical analysis. Data was shown as mean ± the standard deviation (SD). The signi cant differences between groups were estimated by One-Way ANOVA. P < 0.05 was considered as statistically signi cant.
8
Transwell Assay for Cell Invasion and Migration
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Invasion and migration of HCC cells was determined using Transwell assays. As for cell migration, 24 hours after transfection, a total of 2 × 10 4 cells were seeded into the apical chamber supplemented with FBS-free RMPI-1640, while each basolateral chamber was loaded with 600 µL DMEM with 10% FBS. Then Transwells were placed in an incubator at 37°C for 24 hours. The cells in the apical chamber and on the upper surface were wiped out by cotton swabs. The migrated cells were xed in 4% paraformaldehyde (Beyotime Biotechnology Co., Ltd., Shanghai, China) for 20 minutes and then stained with 0.5% crystal violet (Beyotime) for 15 minutes. The invasion of cells was determined in a similar manner with the apical chambers pre-coated with Martrigel (BD-Biosciences, CA, USA) before cell seeding. The number of invaded and migrated cells were observed and counted under an IX81 microscope (Olympus, Tokyo, Japan) with 5 elds randomly selected and the average value was calculated.
9
Transwell Invasion Assay for Cell Migration
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The Transwell invasion assay was detected as previously described [16] . Brie y, the upper chambers (Corning, USA) were coated with diluted Martrigel (BD, USA). Afterwards, the SW620 and HT29 cells were seeded into the upper chambers with serum-free DMEM, then the Transwell chambers were incubated in wells lled with DMEM containing 10% FBS. After 24 h of incubation at 37 °C, the cells in the inner chambers and the remaining Matrigel were removed with cotton swabs, while the cells on the outside surface of the lower chambers were xed using 4% PFA and stained with 0.1% crystal violet for 15 min. After washing twice with PBS, the cell numbers in the ve randomly chosen elds were calculated under a microscope.
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