CytoSelect™ 24-Well Wound Healing Assay

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	- Culture medium containing 5% FBS supplemented with 10 ng/mL VEGF. Cells are allowed to migrate for 4h.

Protocol tips
- Culture medium containing 5% FBS supplemented with 10 ng/mL VEGF. Cells are allowed to migrate for 4h.

Publication protocol

Migration Analysis by Wound Healing and Transwell Assay. HUVECs migration was evaluated by both the CytoSelect24-well Wound Healing Assay and Transwell Migration Assay. A CytoSelect Wound Healing Assay kit (CBA-120-5) purchased from Cell Biolabs (San Diego) was used for this assay. HUVECs suspended in culture medium containing 5 FBS were seeded at 10,000 cells per well on 24-well culture dishes precoated with 0.1% gelatin (Sigma),and then put the CytoSelect TM Wound Healing Insert into the plate wells. After overnight incubation, the inserts were removed from the well to generate a 0.9mm “Wound field.” The cells were incubated with 20𝜇g/mL mitomycin C for 1h to deactivate HUVEC proliferation After that,the cells were washed with PBS to remove debris and incubated with or without VEGF and different concentrations of vitexicarpin. After 12h of incubation, images were taken by a phase contrast microscope (Olympus) and the wound size was determined [30]. The chemotactic motility of HUVECs was assayed using Transwell chamber with 6.5mm diameter non coated filter membrane (pore size, 8𝜇m; Corning, Inc.). Endothelial cell medium with 0.1% FBS supplemented with 10ng/mL VEGF was used as chemoattractant in the bottom chamber, and 5 × 10 4 HUVECs pretreated with mitomycin C in 200𝜇 LECM (0.1%FBS) with different concentrations of vitexicarpin were seeded in the top chamber. The cells were migrated for 4h and the non migrated cells on the top surface of the membrane were removed by wiping with a cotton swab. The migrated cells on the lower side of the membrane were fixed with cold 4% paraformaldehyde for 1h and then stained with hematoxylin and eosin (H&E).Chemotaxis was quantified by manually counting the migrated cells with inverted microscope at ×200 magnification. Four independent areas were counted for each assay.


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Manufacturer protocol

Download the product protocol from Cell Biolabs for CytoSelect™ 24-Well Wound Healing Assay below.

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