Cell Comb™ Scratch Assay

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- Scratched monolayer is washed with Dulbecco’s phosphate-buffered saline to remove any detached cells.	- Post-wounding images are captured every 6h and between imaging sessions cells are returned to 37 °C, 5% CO2

Protocol tips
- Scratched monolayer is washed with Dulbecco’s phosphate-buffered saline to remove any detached cells.
Downstream tips
- Post-wounding images are captured every 6h and between imaging sessions cells are returned to 37 °C, 5% CO2

Publication protocol

The migration ability of endothelial cells was monitored by a scratch wound assay using the four treatment groups as mentioned previously. Twenty-four hours after transfection a confluent monolayer was observed. Cells were then treated with mitomycin C for 2 h to inhibit cell proliferation and washed extensively. The medium was removed, and cells were wounded by scraping with disposable cell combs (cell comb scratch assay kit, catalog No. 17-10191, Merck Millipore). A “cell-free” area with a width of ∼700 μm was created. Cells were immediately washed with DPBS to remove any detached cells from the wound area. Phase contrast images were captured in multiple fields immediately after scratching the cell monolayer (t = 0), and every 6 h until 24 h post-wounding (t = 24) using a Leica microscope. Cells are returned to 37 °C, 5% CO2 between imaging sessions. The length of the wound filled by the migrating cells were measured using Leica Application Suite X (LAS X) software (Leica Microsystems). The percentage of wound closure was calculated based on the equation ((Li − Lt)/Li) × 100%, where Li is the length of wound at t = 0 h and Lt is the length of wound following t hours of culture.

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Manufacturer protocol

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