Cells, as the smallest independent units of structure and function in living organisms, have always been the cornerstone of life science research and occupy a central position in the journey to explore the mysteries of life:

1、Scientific Research:The dynamic processes of cell proliferation, differentiation, signal transduction, and metabolic regulation provide key targets for understanding physiological mechanisms and disease occurrence, making them core tools for revealing the processes of life;

2、Drug Development: Cell models are the core carriers for high-throughput screening, toxicity assessment, and pharmacodynamic validation, with cell activity assays throughout the process from early compound screening to preclinical research. With the innovation of gene editing (such as CRISPR) and cell therapy technologies, cells have become a cutting-edge "drug" for conquering diseases;

3、Production Field: Cells become miniature biofactories, efficiently producing antibodies, proteins, peptides, and other research reagents and drugs for researchers, driving the transformation of life science research and industry.

"How is the growth of the cells I cultured?" This is the primary question researchers ask when cells are exposed to certain substances or influencing factors during experiments. This question can be answered by detecting various indicators of cell activity. Next, I will take you through a comprehensive understanding of the three major categories (a total of six methods) of cell activity assays to help you choose the right method and quickly complete cell viability testing.

1.Star Method, Measuring Cell Activity Based on ATP Content in Cells

The content of ATP (adenosine triphosphate) within cells is a direct indicator of cellular energy metabolism. The ATP bioluminescence method assesses cell activity by measuring the level of ATP within cells. This method is easy to operate, with the entire process simplified into "addition-incubation-reading" three steps, and it features high sensitivity and a wide dynamic range, making it particularly suitable for experiments requiring high throughput and high sensitivity detection.

Schematic Diagram of Cell Viability Assay Using Luciferase Method

2.Compatible with CTG-L reagent, it measures cell viability based on the protease activity of living cells using a non-lytic fluorescence method.

For cell viability assays, there is sometimes a need for an internal reference, which gives rise to the demand for multiplex analysis. This involves conducting multiple analytical methods on the same sample simultaneously, with the requirement that the analytical methods do not interfere with each other and have different detection mechanisms. This allows for correlation and comparison, thereby eliminating data discrepancies caused by experimental factors. The principle of this reagent is to detect a conserved protease within living cells, whose activity is only related to intact living cells. An active substrate that can penetrate the cell membrane enters the living cells and is cleaved by the living cell proteases, generating a fluorescence signal. The intensity of the signal is directly proportional to the number of living cells. When the integrity of the cell membrane is lost and leaks into the surrounding culture medium, the activity of this living cell protease will disappear due to the different environment from within the cell. This reagent has a different detection principle and method from the CTG-L series luciferase reagent kits, and can detect earlier, minor cellular damage. The two detection reagents are compatible and can be used for multiplex detection. This method is simple to operate, with the experiment completed in just three steps: "addition-incubation-reading," making it suitable for high-throughput detection.

Schematic Diagram of Live Cell Protease Activity Assay

3.Traditional Classic Method, Based on the Reductive Activity of Mitochondrial Dehydrogenases, to Measure Cell Viability

The cell viability assay based on mitochondrial dehydrogenase activity primarily relies on the crucial role of these enzymes in cellular metabolism. Located in the inner mitochondrial membrane, these enzymes catalyze the dehydrogenation of substrates (usually compounds involved in redox reactions), transferring electrons to specific electron acceptors (such as the chromogenic substrate in MTT reagent).

When cells are viable, mitochondrial function is normal, and dehydrogenase activity is high, efficiently reducing the substrate and causing a color change (such as the reduction of MTT to purple crystalline formazan). Conversely, when cells lose viability or activity decreases, mitochondrial function is impaired, dehydrogenase activity decreases, the extent of substrate reduction is reduced, and the degree of color change is also diminished.

Due to different detection substrates, these methods can be further divided into:
① MTT method: Requires pre-mixing of two components before use. The working solution can be catalyzed by mitochondrial dehydrogenase to produce non-water-soluble blue-purple crystalline formazan. The product needs to be dissolved by changing the solution, and the absorbance at 570 nm is measured;
② MTS method, an upgraded product of MTT, requires pre-mixing of two components before use. The working solution can be catalyzed by mitochondrial dehydrogenase to produce water-soluble formazan products, eliminating the need to change the solution, making it more convenient, with the absorbance measured at 490 nm;
③ CCK-8 method, an upgraded product of MTT, can be directly added to cell samples without the need to pre-mix various components. It offers rapid detection and very low toxicity. Its effective substrate WST-8 can be reduced by dehydrogenases within mitochondria to form an orange-yellow formazan dye in the presence of an electron coupling reagent, eliminating the need to change the solution, making it more convenient, with the absorbance measured at 450 nm.
④ Alamar Blue method, with the main component resazurin, is a cell membrane-permeable, non-toxic, and weakly blue fluorescent indicator that serves as an alternative to MTT. The oxidized form of resazurin is purple-blue and essentially non-fluorescent, while its reduced product, resorufin, turns pink and highly fluorescent. The fluorescence intensity produced is directly proportional to the number of respiring viable cells. The color change of Alamar Blue can be detected by a standard spectrophotometer by measuring absorbance changes, with a detection wavelength of 570 nm and a reference wavelength of 600 nm; the fluorescence change of Alamar Blue can be detected by a fluorescence spectrophotometer, with an excitation wavelength between 530~560 nm and an emission wavelength of 590 nm.

Schematic Diagram of Cell Viability Assay Based on Mitochondrial Dehydrogenase Activity

These are the three main methods currently used to detect cell viability. I have also summarized the characteristics of these methods here to make it easier for everyone to choose based on actual needs.

That's all for today's presentation. There will be a summary of cell apoptosis and cytotoxicity detection methods coming up, so stay tuned~