The wound healing experiment
The basic procedure to perform a wound-healing experiment is quite straight forward. An artificial cell-free gap is generated in a 100% confluent cell monolayer and the ability of cells to close this gap is monitored.
Many parameters need to be considered and standardized in order to get reliable and reproducible results. The following sections will cover some general aspects about monitoring cells and demonstrate how PAULA can help simplify the workflow tasks.
Suitable contrast methods
In a standard brightfield microscope setup, most cell monolayers are relatively hard to detect or even "invisible". With phase contrast, the cell “shape” is highlighted and allowing the cells to be easily detected. However, the setup for a manual microscope using phase contrast may be cumbersome and time-consuming, especially for untrained users. In general, fluorescence can be used to monitor additional factors of migration. However, it should not be used as a standard contrasting method, because photobleaching and phototoxicity can alter migration significantly.
- PAULA offers alignment-freephase contrast where no fine-tuning is needed.
- Phase contrast can be activated by just one click.
- Fluorescence contrast (red and green emission, e.g., GFP and dsRed labelled cells) can be added if needed.
Wound healing assay with a 100% confluent cell layer
Finding the right point in time matters. In most cases, wound healing assays are performed with a 100% confluent cell layer. To keep cells growing once they have reached 100% confluence can induce cell detachments and even inhibition of neighboring cells. Therefore, finding the right point in time to start the wound healing assay is critical for performing successful experiments yielding meaningful results.
- PAULA informs you via e-mail when your cells have reached the desired confluence.
- You no longer need to go to the cell culture lab and check the cells manually.
- You can find the perfect moment to start the wound-healing experiment.
Optimal environmental conditions are crucial
Wound healing assays are often performed using a so called “dead end” analysis. The cell flasks have to be removed from the incubator and the cells checked manually to determine when the gap is completely closed. This approach is time consuming and not really accurate. Alternatively, the cell flask can be kept on a microscope stage equipped with a climate chamber. In principle, that works fine, but fluctuations in temperature and CO2 cannot be completely excluded. So why not keep the cells where they are most likely to do well? That would mean in an incubator with the most optimized environmental conditions possible for cell culture.
- PAULA can be placed inside a regular cell culture incubator where cells are kept under optimal conditions.
- The focus is stable over days - no readjustment needed.
- PAULA acquires time-lapse recordings and calculates instantly the current migration rate and percent of gap closure, as well as the estimated time to a 50% closure.
- The cell status and results can be constantly monitored from anywhere.
Consistent gap size is important
In many cases, the cell-free gap in the monolayer is created manually with a pipette tip. The scratch size largely depends on the pressure applied to the monolayer surface. However, to get reproducible results, a consistent scratch size is important.
- Determine the exact cell-gap width with PAULA’s integrated ruler.
- Find more easily comparable gap sizes between different flasks.
- PAULA can also be used for insert-induced cell-free gaps, e.g., from ibidi.
Other important parameters for reproducibility
In addition to gap size and environmental conditions, there are other factors that can influence the reproducibility of wound healing assays. For example, technical parameters like image and acquisition settings and system calibration, as well as, specimen conditions like the passage number of the cells. PAULA monitors both types of parameters.
- PAULA can record the cells’ passage number. This way you know at all times if you are really comparing similar cell states (apples with apples or oranges).
- Recall all your personal settings used for a particular specimen, including the fine-tuning of parameters such as for lighting.
- Extra calibration of the system is not necessary.
Easy setup of the wound healing assay
If the setup of a wound-healing experiment is simple and easy to repeat many times, then the results are much more likely to be reproducible. See for yourself how easy and self-explanatory the setup of the wound healing assay is with PAULA. Only 3 steps are necessary to start a successful wound-healing experiment.
- Define your specimen
- Define t-lapse
- Define region of interest
The results count
At the end of the day, the results are what really count.
- PAULA provides a graphical display of the gap-closure values and migration rates over time.
- Points in time used for the gap and rate calculations can be arbitrarily defined, so that the effects of dying cells at the borders of the scratch can be eliminated.
- Time to 50% and 100% gap closure are first estimated and then the actual values calculated if reached.
- All data can be displayed in a list and exported into Excel files.