Cell-based assays have been long used to foretell how a drug would affect an organism and have emerged as a great substitute biological drug discovery method that complements other technologies as well. While earlier cell-based assays were used more for secondary screening, gradually it is being promoted to the first.

The cell-based assay has proved to be extremely valuable for vetting various kinds of compounds and is increasingly used in many research studies for evaluating and identifying pathways in the whole cell as well as collect data about biological targets.

To eliminate variability and noise from the cell-based assay screening and improve the performance, you will require consideration, understanding, and manipulations. Here are some factors to consider before you choose a cell-based assay to ensure that the test runs successfully.

Tips for Performing a Successful Cell-Based Assay

Choosing the Right Cell Types:

One of the key factors that affect the outcome of a cell-based assay is the kind of cell type chosen for the experiment. Experimenting with Cell lines and primary cells results in different types of biomarkers that can impact the assay results. While primary cells like hepatocytes and HUVECs are perfect for conducting in-vitro studies, it is difficult to grow these cells and transfect them.

On the other hand, cell lines are known for their high metabolic rate which is why they are mostly used in performing cell vitality assays that tend to use a metabolite. Similarly, one of the holdbacks of using primary cells in that it has a higher rate of spontaneous death. So, you have to be careful about the kind of cell type you choose for the assay.

The Edge Effect:

One of the known factors behind the decline in assay performance is edge effects. Edge effect during an assay can lead to high plate rejection during the screen runs. Some of the steps that you can take to avoid the edge effect are:

Ensure to keeps copies of the samples, monitor the plate order, position, and timing in the incubator while maintaining an even temperature.

Another way to reduce or eliminate the edge effect is to incubate new seeded plates at room temperature before putting them in the incubator.

Understanding the Death Process of a Cell:

As stated before, choosing the right cell type to perform the assay is very important and to choose the right cell type it is essential to understand cell apoptosis or in layman terms the death process of a cell. This helps you determine if the cell for the assay and help you come up with the right experimental design and opt for the right endpoint selection.

Avoid or Eliminate Contamination:

Common contaminates like foreign cells, mold, and bacteria can lead to irreproducible and inconsistent cell-based assay results. Before performing the assay ensure that cover the plates properly before incubating them. Apart from also clean the pipettes, media bottles and other equipment used in the assay properly with alcohol before performing the cell-based assay.

Avoid Pipetting Errors:

Making mistakes while pipetting is quite common and results in false cytotoxicity, poor health, and cell death. You must carefully and evenly distribute the cells from one well to another or during the preparation of frozen aliquots. Ensure to calibrate the tool to prevent errors during pipetting while handling experimental data.

Regulate Carbon Dioxide Level and Temperature:

Maintaining the CO2 levels and temperature is critical in keeping the cell vitality intact while performing cell-based assays. If the cells are exposed to room temperature before incubation, it will result in a decrease of signal to background ratios. Similarly, high CO2 concentration during incubation will negatively affect cell activity. Such small changes in the surrounding environment can adversely impact the assay result. Therefore, ensure to eliminate such factors while performing cell-based assays.

Use Assay Controls:

One of the most important steps in experimental design is to include control, to help determine if the assay is working properly. Apart from this function, control samples also enable the researchers to compare the data and study the variables in the samples. Many types of controls are considered while performing cell-based assay like negative and positive controls, no cell, untreated cell control. However, no matter what type of control you use, it will provide you with a reference point to help you analyze the data obtained from the assay.

Consistent Cell Density:

If you want to accurately determine the assay range, sensitivity, and collect the relevant data, then ensure that the samples have uniform cell density. Less than optimal cell density can lead to higher detection sensitivity while high cell density will result in higher background signals. The best way to maintain the optimal cell density is to generate a standard curve of different types of cell numbers for better cytotoxicity and viability. Generating a standard curve will give you a precise idea of the cell density that you need before adding the test compound in the cell-based assay.

These are some of the tips that you can follow to perform a successful cell-based assay. If you have anything more to add, do let us know in the comments below.