IMAGEJ SOFTWARE WOUND HEALING ASSAY MANUAL
IMAGEJ SOFTWARE WOUND HEALING ASSAY SERIES

Automation also has greater functionality for example, it can be used to determine the experimental endpoints and generate kinetic, functional, and quantitative measurements of living cells (see Figure 2). Aspects of acquisition that can be automated include image capturing, point visiting, and environmental control.

IMAGEJ SOFTWARE WOUND HEALING ASSAY MANUAL

The disadvantages experienced with manual acquisition can largely be overcome using automation that allows for live-cell imaging. Accurate measurements can be manually acquired using a digital camera fitted to the microscope however, this process is time-consuming, and maintaining the same field-of-view along each gap can be difficult. The migration images can then be used to collect measurements or be visually scored. These time points should be collected within 24 hours of the experiment being started to minimize the confounding effects of cell replication on gap closure.

IMAGEJ SOFTWARE WOUND HEALING ASSAY SERIES

Once the microscope is set up, a series of time-lapse images (snapshot method) can be acquired as cells migrate into the cell-free gap. Cell migration is best viewed using phase-contrast imaging rather than fluorescence, and the wound area in the field-of-view should be maximized using the objective lens. With a cell-free gap prepared, optical microscopy can be used to observe cells migrating into the wound area. Data acquisition вЂ“ scratch images for time-lapse Ensuring reproducibility is important for the following data acquisition step. Modifications to the wound healing assay that make use of automation can increase both throughput and improve reproducibility. This reproducibility can be improved with the use of commercial tools for making uniform gaps. Drawbacks with the manual method of wounding include low throughput and well-to-well variation of the gap width. The preparation of gaps can be performed manually or be automated. Methods that avoid cell damage altogether, such as physical exclusion, have also been described. Have been used to clear cells from fluid channels,. In microfluidic wound healing assays, chemical and enzymatic treatments Alternatives to mechanical damage can include thermal, electrical, and optical wounding. The most frequently used method is to wound the monolayer by mechanical scratching (scratch wound) or stamping.

Scratch-makingДЄfter the cells have become confluent, the next step is to make a cell-free gap in the monolayer. Careful optimization is necessary as any changes to the medium can result in unpredictable time- and cell-type-dependent effects that can interfere in the assay. Proliferation inhibitors, such as mitomycin C, can also be added to the medium. Decreasing the concentration of serum (serum starvation) is the most common change. If this occurs, the cell medium can be optimized to reduce cell proliferation. Cell proliferation can compete with cell migration to fill the gap made during the assay.

Дepending on the cell type used in the assay, cell proliferation can be a confounding factor.

Studies have also examined the migration of vascular smooth muscle cells. Most often, epithelial and endothelial cells are used to produce the monolayer, and in particular, cell types capable of sheet migration. This monolayer represents the in vivoД¬onditions of the tissue before wounding such as, an intact epithelium. The first step of the assay is to culture a confluent cell monolayer. Wound healing assay protocol Culture preparation The technique reproduces wounding by creating a gap in a confluent cell monolayer and consists of four main steps that will be elaborated upon below (see Figure 1 for an overview). While this assay does not recapitulate the exact conditions of a wound, it can be successfully applied to model and investigate cell movement in a controlled in vitro environment. The wound healing assay is a simple and inexpensive method. Wound healing experiments with these CytoSMART devices: full-plate scanner Omni,ДЇluorescence cell imager Lux3 FL, mini live-cell imagers Lux2 and Lux3 BR. Three-dimensional (3D) wound healing assays are beyond the scope of this article and have been succinctly covered by Stamm et al. This method was one of the first to be developed for the study of cell migration and measures the rate at which cells, in a cell monolayer, migrate to fill a cell-free gap. The wound-healing assay, also known as the scratch assay, is an established two-dimensional (2D) technique that can be used to investigate collective migration and wound healing in vitro. on In vitro wound-healing assay also known as the scratch assay

Imagej software wound healing assay