The migration capacity of MSCs was determined by a standard transwell migration assay using transwell chambers with 8 μm pore size (Corning Costar, USA). After transfection by rHDL/PEI-LA/pTRAIL nanoparticles or PEI-LA/pTRAIL complexes as described in Section 2.8, the treated MSCs were trypsinized and suspended in 200 μL of culture media to plate in the upper chamber at the density of 2 × 104 cells/mL. The untreated MSCs were set as positive control. Then B16F10 cells with same density were seeded in the lower chamber of transwell plate (the total volume was 500 μL) and the untreated MSCs in the upper chamber and culture medium without B16F10 cells in the lower chamber were acted as negative control. MSCs remaining on the upper face of the filters were removed by cotton swabs after incubated for 24 h at 37 °C, and the migrating MSCs were fixed with 4% paraformaldehyde PBS solution and stained with crystal violet. Cells on the insert membrane were observed by a transmitted-light microscopy (EVOS XL, ThermoFisher Scientific, USA). Results were expressed as the average number of migrating cells for three replicates from five random fields.
Transwell chambers with 8
Manufactured by Corning
Sourced in United States
Transwell chambers with 8 are a type of cell culture insert used for in vitro studies. These chambers consist of a porous membrane that separates the upper and lower compartments, allowing for the study of cell migration, permeability, and other cell-based assays. The core function of Transwell chambers is to provide a controlled and standardized environment for the cultivation and analysis of cells.
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4 protocols using transwell chambers with 8
1
Transwell Assay for MSC Migration
2
Transwell Migration Assay with JNK-IN-8
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Twenty‐four‐well Transwell chambers with 8‐mm pore size (Corning Costar) were used to perform migration assay. The upper chamber was inoculated with 5 × 104 TPC2‐4 cell suspension (control, TNFα‐induced cells treated with or without JNK‐IN‐8) re‐suspended with DMEM/F12. The lower chamber was filled with 5% foetal bovine serum contained‐DMEM/F12 medium. Cells in the upper chamber were carefully removed 24 h later. The migrated and invaded cells in lower chamber medium were imaged and calculated.
3
Renca Cell Migration and Invasion Assays
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For the migration assay, Renca cells were plated in six-well plates at a density of 3 × 105 and incubated with medium alone or serial dilutions of lycorine hydrochloride (0, 1, 2.5, 5, and 10 μM) for 24 h. Renca cells were resuspended in serum-free medium and seeded into trans-well chambers with 8-μm diameter pore size polycarbonate membranes (Corning, NY, USA). Medium containing 30% fetal bovine serum was used in the lower chamber as an attractant. For the invasion assay, trans-well chambers were coated with Corning Matrigel Matrix (Corning, NY, USA). After 12 h, cells on the upper chamber surface were removed using cotton swabs. The migrated or invaded cells in the lower surface were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The total numbers of cells were captured and analyzed from 10 different fields with the Olympus IX2 microscope.
4
Cell Migration and Invasion Assays
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Cell migration capacities were evaluated via wound-healing assays. The cells were placed in 35-mm plates, and once they reached 90% coverage, a scratch was made using a pipette tip. After being rinsed with PBS, the cells were subsequently incubated in a serum-free medium. The photographs were taken at the specified time intervals to record the scratch closure using a microscope (Olympus IX73).
Cell invasion capacities were evaluated via the transwell assays. Transwell chambers with 8 μm pores (Corning) were pre-coated with Matrigel (BD Biosciences). Cells in serum-free medium were plated to the upper chamber (5 × 104 cells/well) and incubated with medium containing 10% FBS for 24 h. Then, the penetrated cells on the inserts were fixed with 4% paraformaldehyde and stained with Crystal Violet (Beyotime). The cells that invaded to the lower surface of the membrane were photographed with a light microscope.
Cell invasion capacities were evaluated via the transwell assays. Transwell chambers with 8 μm pores (Corning) were pre-coated with Matrigel (BD Biosciences). Cells in serum-free medium were plated to the upper chamber (5 × 104 cells/well) and incubated with medium containing 10% FBS for 24 h. Then, the penetrated cells on the inserts were fixed with 4% paraformaldehyde and stained with Crystal Violet (Beyotime). The cells that invaded to the lower surface of the membrane were photographed with a light microscope.
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