Cell migration assay was performed using a Boyden chamber (6.5 µm diameter, 8 µm pore size, 24 well-plate; Cell Biolabs). Transfected cells were incubated with serum-free MEM (starvation medium) for six hours and seeded into a transwell filter in starvation medium. MEM containing 10% FBS was placed into the lower chamber, and cells were allowed to migrate for 24 h. Unmigrated cells on the upper surface of the transwell filter were removed with a cotton swab. Migratory cells in the lower chamber were stained with 0.4% Giemsa. Migrated cells were counted in five different random fields per well under a light microscope.
Boyden chamber
Manufactured by Cell Biolabs
Sourced in United States
The Boyden chamber is a laboratory equipment used to study cell migration and invasion. It consists of two compartments separated by a porous membrane. Cells are placed in the upper compartment, and a chemoattractant or other stimuli are added to the lower compartment. Cells that migrate through the porous membrane can be quantified, providing a measure of their migratory or invasive potential.
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Lab products found in correlation
Protocol hema 3 staining kit, by Thermo Fisher Scientific (3 mentions)
Formaldehyde, by Merck Group (2 mentions)
Crystal violet, by Merck Group (1 mentions)
Boyden chamber, by Merck Group (1 mentions)
Celltiter 96 aqueous cell proliferation assay kit, by Promega (1 mentions)
Ix73 fl, by Olympus (1 mentions)
10 protocols using boyden chamber
1
Cell Migration Assay using Boyden Chamber
2
Migration and Apoptosis Assays
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Migration assay was performed using the Boyden chamber (Cell Biolabs Inc. US) with transwell inserts of 8 μm pore size as described in our previous study [17 (link)]. The migrated cells from five random fields were counted under the microscope (Zeiss, Germany). Apoptosis assay was assessed by flow cytometry of Annexin V staining as described in our previous study [13 (link)]. The treatment duration for migration and apoptosis were 8 h and 72 h, respectively.
3
Boyden Chamber Migration Assay
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Migration assay was performed using the Boyden chamber (Cell Biolabs) as described in our previous studies [17 (link), 18 (link)]. Briefly, 1000 cells were added onto upper chamber, and drugs were added onto the lower chamber. After 12 h incubation, the non-migratory cells on the upper surface of the insert were removed with cotton bud. Migratory cells on the lower surface of inserts were fixed with 4% formaldehyde (Sigma) and stained with 0.4% Giemsa. The photos were taken under microscope and migrated cells from five fields (up, low, left, right and centre) were counted for quantification.
4
Neutrophil Migration Assay with Synaptamide
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Neutrophil migration was measured by the Boyden chamber (Cell Biolabs, San Diego, CA). The cells were resuspended in serum-free DMEM containing synaptamide (10 nM) and added to the upper compartment of the chamber. The lower compartment was filled with 30 nM LTB4 and 10 ng/mL CCL2 in 150 μL DMEM. The two compartments were separated by 3 μm polycarbonate membrane. After 30-min incubation with LTB4/CCL2 and synaptamide, cell migration to the lower chamber was detected using the CytoSelectTM migration assay kit (Cell Biolabs, San Diego, CA) according to the manufacturer’s instructions.
5
Boyden Chamber Cell Migration Assay
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We performed migration assay using the Boyden chamber (Cell Biolabs) using the same protocol as described in our previous study [19 (link)]. Briefly, cells were placed onto upper chamber and drugs were added to lower chamber. After 8 h incubation in a tissue-culture incubator, the migrated cells were counted under microscope.
6
Evaluating Cell Migration and Invasion
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For wound-healing assays, PCa cells in six-well culture dishes were allowed to grow to 80~90% confluence, and a sterilized tip was utilized to introduce a scratch “wound” with the same width on the bottom of the dishes. We generally made two scratch wounds per well as technical replicates, and wound closure under multiple microscopic views per well was recorded. Images were captured at 0 and 20–48 h after the wounding depending on cell types. Data shown were representative of three independent repeats. Moreover, cell migration and invasion assays were performed using Boyden chambers (CellBiolabs, San Diego, CA) according to manufacturer’s instructions. Briefly, PCa cells were loaded into the chambers and cultured in media with or without varying concentrations of E7107 for 1–2 days, and results were visualized by PROTOCOL™ Hema 3 staining kit (Fisher Scientific, Pittsburgh, PA). Images of the membranes were captured by Olympus IX71. Data were quantified based on the cell number counting of at least five 10× images.
7
Boyden Chamber Assay for Cell Migration
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To further assess the migration of MDA-231 cells, we also performed the Boyden chamber assay. Boyden chambers (Cell Biolabs Inc., San Diego, CA, USA) were pre-incubated for 30 min at 37 °C in 24-well plates containing 600 μl of culture medium and FBS (1 : 1). Cells, previously starved for 24 h, were added on top of the chambers, treated with growth factors and/or drugs at different oxygen levels (pO2 21% or 1%) and allowed to grow and migrate for 24 or 48 h. At the specified end points, the chambers were removed from the plates, fixed and stained in two steps (Eosin+AzurA/AzurB, 2 min each) and photographed. Analysis did not involve counting the number of migrating cells but only assessing visible differences between the treatments. The experiments were repeated twice.
8
Quantifying Prostate Cell Migration
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Cell migration and invasion assays were performed using Boyden chambers (CellBiolabs, San Diego, CA) according to manufacturer's instructions. Briefly, freshly purified basal and luminal cell populations were loaded into the chambers and cultured in media for 2 days, and the results were visualized by PROTOCOL Hema 3 staining kit (Fisher Scientific, Pittsburgh, PA). Images of the membranes were captured by Olympus IX71. Data was quantified based on the cell number counting of at least five × 20 images. To test the response of prostatic basal cells to neural signals (Supplementary Fig. 6b ), primary basal cells were incubated in the chambers in media with or without neural growth factors (20 ng ml−1 of BDNF/GDNF/NGF-β, 500 μM GABA and 0.5 mM db-cAMP).
9
Cell Proliferation and Invasion Assays
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Cell proliferation assays were carried out as described previously13 (link). Briefly, cells were seeded at a density of 2,500 cells per well in 96 well microplates and cultured in standard medium. Viable cell numbers were determined 0, 1, 2, 3 and 6 days after cell plating using CellTiter 96 Aqueous Cell Proliferation assay kit (Promega, Madison, USA). For invasion assay, Boyden Chambers (Cell Biolabs, San Diego, USA) were layered with 2 mg/ml extracellular matrix gel from Engelbreth-Holm-Swarm mouse sarcoma (Sigma-Aldrich, St. Louis, USA) overnight at 37 °C. Cells were seeded at a density of 100,000 per well. Wells were then filled with standard medium. After 48 h, Boyden Chambers were washed twice with PBS, followed with fixation of the cells using 4% formaldehyde (Sigma Aldrich). Subsequently, cells were stained with 1% Crystal Violet (Sigma Aldrich), photographed and counted.
10
Wound Healing and Migration Assays for PCa Cells
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For wound-healing assays, PCa cells in six-well culture dishes were allowed to grow to ~85% confluence, and a sterilized tip was used to introduce a scratch “wound” with the same width on the bottom of the dishes. We generally made two scratch wounds per well as technical replicates, and wound closure under multiple microscopic views per well was recorded. Images were captured at 0 and 20 to 48 hours after the wounding depending on cell types. The data shown were representative of three independent repeats (60 (link)). Alternatively, cell migration and invasion assays were performed using Boyden chambers (CellBiolabs) according to the manufacturer’s instructions. Briefly, PCa cells were loaded into the chambers and cultured in media with or without varying concentrations of indicated chemicals for 1 to 2 days, and results were visualized by PROTOCOL Hema 3 staining kit (Fisher Scientific). Images of the membranes were captured by Olympus IX73FL. Data were quantified on the basis of the cell number counting of at least five to six ×20 images.
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