The invasion capacity of MSCs was analyzed using Matrigel-coated transwell cell culture chambers (8 μm pore size; Sigma-Aldrich). MSCs (1×104 cells) were seeded in the inserts of transwell chambers in serum-free medium, and culture medium with 10% fetal bovine serum was maintained in the lower chamber. All samples were incubated for 48 h at 37°C. In the transwell chambers, cells were stabilized with 4% paraformaldehyde in phosphate-buffered saline (PBS) and stained with 2% crystal violet in 2% ethanol. The lower surface of the transwell chamber contained invasive cells, which were quantified and photographed using a light microscope. Non-invasive cells were discarded with a cotton swab.
Matrigel coated transwell cell culture chambers
Manufactured by Merck Group
Sourced in United States
Matrigel-coated transwell cell culture chambers are laboratory equipment designed for studying cell migration and invasion. They consist of a two-chamber system separated by a porous membrane, with the upper chamber coated with Matrigel, a reconstituted basement membrane extract. This setup allows researchers to assess the ability of cells to migrate through the membrane and Matrigel barrier, which can provide insights into cellular processes such as cancer metastasis, angiogenesis, and immune cell trafficking.
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8 protocols using matrigel coated transwell cell culture chambers
1
Matrigel-Based Invasion Assay for MSCs
2
Exosome-induced Cell Invasion Assay
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Matrigel-coated transwell cell culture chambers (8-μm pore size; Sigma-Aldrich) and serum-free RPMI-1640 or EBM-2 medium were used to assess the invasion of SNU-C5/5FUR, SNU-C5/OXR, and HUVECs. The cells were first treated with SNU-C5/5FUR or SNU-C5/OXR exosomes derived from different conditions for: hypoxic, normoxic, and/or transfected with si-PRNP, and incubated for 72 h at 37 °C, then invasion assay was performed. Cells were stained with 2% crystal violet, and invasive cells were quantified and photographed using a light microscope.
3
Matrigel-Coated Transwell Assay for Cell Migration and Invasion
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Cell migration and invasion were determined with Matrigel-coated Transwell cell culture chambers (8-μm pore size) (EMD Millipore) as described previously [19 (link)]. U2OS cells were seeded (1×104 cells/well) into the upper chamber with serum-free medium. The bottom chamber contained medium with 0.5% DMSO, cariporide, or DMTU + cariporide. After 48 h of incubation, the cells in the upper chamber were removed and the cells that invaded through the membrane were fixed with 70% ethanol and stained with 2% crystal violet in 2% ethanol. Invaded cells in six random microscopic fields (200 × magnification) were counted and all experiments were performed in triplicate.
4
Evaluating hTERT-ADSC.CD Migration Potential
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The migration of hTERT-ADSC.CD towards PC cells was evaluated in Matrigel-coated transwell cell culture chambers with a pore size of 8 μm (Merck Millipore, Billerica, MA, USA). At 24 h prior to commencing the experiment, 1.5 × 104 cells of human prostatic myofibrosblast lineage, WPMY-1, or alternatively, medium, were added to the 24-well plates in the lower well. 105 RT-ADSC and hTERT-ADSC.CD cells, suspended in a serum-free medium were added to the ECMatrix inserts (pore size, 8 Am). PBS was used to rinse the cells in the lower wells. Serum-free medium was then added, and then the wells were placed in an incubator for 48 h for the migration assay. ECMatrix, together with any non-invading cells, was removed from the insert interior. Following 20 min exposure to the stain, photographs were captured microscopically. The abundances of migrated cells within 5 regions of interest were counted, and the mean computed. Images of cells were captured with a camera. An image analyzer system was used to obtain quantitative data (National Institutes of Health [NIH] Image J 1.34, http://rsbweb.nih.gov/ij/ , accessed on 1 December 2023).
5
Quantifying HCC Cell Migration and Invasion
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HCC cell migration was assessed by the wound healing assay. HCC cells were cultured in a tight cell monolayer in six-well plates, and wounded with 200 µL plastic pipette tips. The migrating distance of cells at the wound front was determined in photographs of microscopic fields at 200× magnification. HCC cell invasion was analyzed with Matrigel-coated transwell cell culture chambers (EMD Millipore, Billerica, MA, USA). BEL-7404 cells (5×104) and SMMC-7721 cells (3×104) were plated in the top chamber onto the Matrigel-coated membrane (24-well insert) and allowed to migrate toward serum-containing medium in the lower chamber. The cells were incubated for 24 hours, then fixed with 4% formaldehyde for 15 minutes and stained with 0.5% crystal violet for 15 minutes. The invaded cells were counted at 200× magnification from five different fields.
6
Transwell Assay for Tumor Cell Tropism
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Matrigel-coated transwell cell culture chambers (8-μm pore size; Merck Millipore, Billerica, MA, USA) were used to assess the tropism of hTERT-ADSC.sTRAIL for tumor cells. Medium or 1.5 x 104 WPMY-1, human prostatic myofibroblasts (ATCC, Manassas, VA, USA), were loaded in the lower well of 24-well plates 24 h before the start of the experiment. The hTERT-ADSCs and hTERT-ADSC. sTRAIL (105) cells in serum-free medium were placed on the 8-Am pore-size inserts coated with ECMatrix. The lower wells containing the cells were washed with phosphate-buffered saline (PBS), filled with a serum-free medium, assembled for the migration assay, and incubated for 48 h. The noninvading cells and ECMatrix were wiped away from the inside of the insert, and the cells were stained for 20 min. The invading cells were photographed under a microscope. The results were evaluated by directly counting the number of migrated cells in five fields and calculating the mean.
7
Transwell Invasion Assay Protocol
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The migration and invasion ability of cells were assessed using the Matrigel‐coated Transwell cell culture chambers (8‐μm pore size; Millipore, Billerica, MA, USA). Briefly, 700‐μL complete DMEM medium was added to the lower chamber in a 24‐well plate. Then, cells to be placed on the top were resuspended in the serum‐free DMEM medium, and 200 μL of the resuspended cells solution (1 × 105 cells) was seeded into the upper chamber of each Transwell. After 24‐hour incubation, the cells were fixed with 4% formaldehyde for 10 min at RT and permeabilized by 100% methanol for 2 minutes. After washing with PBS, cells were stained with 10% Giemsa staining solution for 15 minutes at RT. Finally, after noninvasive cells were scraped off with a cotton swab, invasive cells were counted in five randomly selected areas per well (magnification, ×200). Data were obtained in triplicates.
8
Measuring Cell Invasion and Migration
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Cell invasion and migration were respectively analyzed by Matrigel coated transwell cell culture chambers (8 μm pore size, Millipore, Billerica, MA, USA) and the scratch wound-healing motility assay following the protocol of our previous studies [13 (link), 50 (link)]. All experiments were done in triplicate. Mean normalized gene expression ± SE was calculated from independent experiments.
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