Selecting the right methods for your needs
It is almost always most effective to combine several different methods of assessing cell health, as no one method gives a perfect view of cell viability, proliferation, or cell death. Each method has Show more
What you want to measure
Understand what it is you want to measure and define what your endpoint will be. The most appropriate assay will depend on whether you’re looking to determine the number of living versus dead cells, quantify cell damage or understand the underlying mechanisms. Using multiple endpoints may be the best approach.
Many viability assays, such as MTT, don’t measure cell number but overall metabolic activity. Therefore, your result may indicate a change in cell number due to either cell proliferation or cell death, or a combination of the two, as well as a change in metabolism. Depending on what you’re looking to measure, you may need to combine MTT with another assay to be sure.
Knowing the markers of cell viability, cytotoxicity and apoptosis will enable you to select an assay that will help you answer your question.
The model system that you’re using
It is important to identify and consider any aspects of the model system that you’re using that will affect the outcome of your assay. For example, cell lines have a higher metabolic rate than primary cells.
In addition, suspension cells will benefit from an assay that has minimal or no steps that require removal of the media as this will reduce potential cell loss during these wash steps.
Number and type of cells
If you have a lot of samples, this is likely going to affect which assay you choose. Some assays require manual counting, such as the viability assay trypan blue, which is more hands-on and time consuming than MTT, for example, and so is not practical for large numbers of cells.
On the other hand, if you have a low number of cells, you will need an assay with a higher sensitivity.
Whether you’re using cell culture or tissue samples
You sample type will also influence your choice of assays due to factors such as the inability of some reagents to penetrate through multiple layers of cells.
For example, TUNEL assays and proliferation assays such as BrdU are commonly used for tissue analysis, whereas MTT assays are more commonly used in cell culture.
Many of the cell functions being measured with the assays described in this guide, such as metabolism, proliferation and apoptosis are interlinked1 by a complex web of processes, therefore the best approach to selecting assays is to thoroughly understand how they work.
How frequently should I assess cell health?
At a minimum, it is recommended that you assay cell health prior to your experiment and at any critical timepoints, such as cell passaging.
Depending on the experiment you’re doing and the reason why you’re measuring the health of your cells, you may want to assay cell health more often, to provide insight into ongoing changes or identify any issues with your cell culture process. Consider, however, that most assays do not allow for continued culture. Frequent assays could therefore risk using up all your cells, especially slow growing or more precious cultures.
Instrumentation
Available instrumentation will be another consideration when reviewing your assay options. Analysis commonly requires a fluorescence microscope, flow cytometer, or microplate reader.
Most assays described in this guide utilize fluorescence, luminescence or absorbance as endpoints, therefore a well-optimized plate reader capable of detecting the right endpoint is required. It’s also worth considering what kind of plates to use – generally clear plates are recommended for absorbance assays, white for luminescence assays and black for fluorescence assays.