HUVECs were seeded into the upper compartment of transwell inserts of Boyden chambers (24-well plate; Corning, Corning, NY, USA). Conditioned media from HCLE cells were prepared as described above. Bevacizumab (Avastin; Genentech BioOncology, San Francisco, CA, USA) at 60 μg/mL was added to untreated HCLE media as a negative control for the migration assay. We added 500 μL of HCLE conditioned media into the lower compartment of the Boyden chamber. After 5 hours of incubation at 37°C in a humidified 5% CO2 incubator with the conditioned media, the transwell inserts were fixed with 4% paraformaldehyde and stained with 0.2% crystal violet. The inserts were carefully washed with distilled water and left to dry overnight at room temperature. The inserts were imaged and the migrated cells were quantified to determine number of migrated HUVEC cells. At least three independent experiments were performed, and statistical significance was evaluated using Student's t-test.
Boyden chambers 24 well plate
Manufactured by Corning
Sourced in United States
Boyden chambers (24-well plate) are a type of cell migration assay used to study the directional movement of cells in response to a chemical gradient. The chambers consist of an upper and lower compartment separated by a porous membrane, allowing cells to migrate from the upper to the lower compartment.
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2 protocols using boyden chambers 24 well plate
1
HUVEC Migration Assay with HCLE Conditioned Media
2
Transwell Assay for Cell Migration
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HUVECs were seeded into the top compartment of the transwell insert of Boyden chambers (24-well plate, Corning). 300 µl of conditioned media from WT, OE, KD, and shCTRL cells were added to the bottom compartment of the chambers. Chambers were placed in a humidified incubator with 5% CO2 at 37 °C for 4 h. The transwell inserts were gently washed with 1× DPBS (Corning), fixed with 4% paraformaldehyde, and stained with 0.2% crystal violet. The inserts were then washed with distilled water and left to dry overnight at room temperature. Lastly, the inserts were imaged using an Olympus BX-51 (Waltham, MA) microscope and migrated cells were quantified. Three independent experiments were conducted, and statistical significance was established using Student’s t-test (unpaired, two-tailed).
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