A Boyden chamber assay (Cell Biolabs INC, San Diego, CA) was performed as per manufacturer's instructions using an FBS gradient. A positive control used cells seeded into the lower chamber, and the negative control was without the FBS gradient (no FBS in lower or upper chamber). To inhibit proliferation 10 μg/ml mitomycin C was added to the upper chamber.
Boyden chamber assay
Manufactured by Cell Biolabs
Sourced in United States
The Boyden chamber assay is a well-established laboratory technique used to study cell migration and invasion. It consists of a two-chamber system separated by a porous membrane, allowing cells to migrate from the upper chamber to the lower chamber in response to chemical stimuli. The assay provides a quantitative measurement of cell migration and invasion, which can be useful for various applications in cell biology, cancer research, and drug discovery.
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5 protocols using boyden chamber assay
1
Boyden Chamber Cell Migration Assay
2
Cell Proliferation, Apoptosis, and Migration
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Cell proliferation was carried out using the BrdU Cell Proliferation Assay kit (Cell Signaling, US). Cell apoptosis was determined by firstly staining apoptotic cells using annexin V-FITC and propidium iodide (PI) apoptosis kit (Sigma-Aldrich, USA), followed by analyzing cells using flow cytometry (Beckman Coulter, USA). Cell migration was determined using the Boyden chamber assay (Cell Biolabs Inc, USA). Briefly, cells together with zileuton were seeded in the top of the insert in serum-free media. Media with serum was placed in the well below. Nonmigratory cells were removed with a cotton swab. Migratory cells move through the pores below were stained with Giemsa (Sigma) and counted under microscope. Specific condition for each experiment is described in the figure legends.
3
Mesenchymal Stromal Cell Migration Assay
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Mesenchymal stromal cell migration was measured using the Boyden chamber assay (Cell Biolabs, Inc., San Diego, California) in a 24‐well format with 8 μm pore size according to the manufacturer's instructions. In brief, 5 × 104 cells in 300 μL serum‐free medium were seeded in the upper compartment (the insert) and then allowed to migrate through the pores of the membrane into the lower compartment. After an appropriate incubation time in a cell culture incubator, migratory cells on the bottom of the polycarbonate membrane were stained and quantified in a fluorescence plate reader.
4
Comprehensive Cell Proliferation and Migration Assay
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Cell proliferation was measured using BrdU Cell Proliferation ELISA Assay Kit (Cell Signaling). Cell apoptosis was assessed by measuring cytosolic oligonucleosome‐bound DNA using a Cell Death ELISA kit (Roche). The absorbance at 450 nm was measured on a microplate reader (SpectraMax). Migration was assessed using Boyden Chamber assay (Cell Biolabs). In brief, cells were seeded on the topside of the insert in serum‐free media and a medium with 10% serum was placed in the well below. After 6–8 h incubation in a cell culture incubator, migratory cells moved through the pores toward the serum below. These were then were fixed with 10% formalin, stained with Giemsa, and counted under a light microscope.
5
Quantifying HUVECs Migration Capacity
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The migration capacity of HUVECs in response to CD34− cells was measured using the Boyden chamber assay (Cell Biolabs, San Diego, CA). Cells (3×105) were placed in the upper part of a Boyden chamber. The chamber was placed in a 24-well culture dish (Thermo SCIENTIFIC) containing EBM-2, EGM-2, or EGM-2 containing CD34− cells for 24 h at 37°C. For quantification, cells migrating to the lower chamber were counted in 5 random microscopic fields using a hemocytometer (Marienfeld Superior, Lauda-Königshofen, Germany). Results are expressed as the number of migrated cells per 300,000 cells added to the Boyden chamber.
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