As the basic unit of life, the vitality of cells directly reflects an organism’s health status and disease progression. Assessing cell viability is like conducting a comprehensive “physical examination” for cells. It helps researchers and clinicians gain deeper insights into physiological functions, drug effects, and mechanisms of disease development.
Today, let’s delve into the world of cell proliferation, cytotoxicity, and viability assays, exploring some of the most commonly used and important detection methods.
1. Trypan Blue Staining: A Direct Assessment of Cell Death
Trypan blue is a large, anionic dye. The intact membrane of live cells exhibits selective permeability and can exclude the dye. In contrast, dead cells lose membrane integrity, allowing trypan blue to enter and stain the cells blue. Thus, blue-stained cells are considered non-viable, while unstained cells are viable.
Trypan blue exclusion is widely used in quality control during cell culture, such as after passaging or thawing. It is also applied in cytotoxicity assays for preliminary screening of drugs or chemicals with evident toxicity. However, this method only distinguishes live from dead cells and lacks quantitative data on cell viability. It may not accurately assess mildly damaged or early-stage apoptotic cells.
2. MTT Assay: A Classic Method for Assessing Cell Viability
The MTT assay is a widely used, classic method in cell viability analysis. It is based on the principle that MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), a yellow compound, is reduced by mitochondrial succinate dehydrogenase in viable cells to form insoluble blue-purple formazan crystals. These crystals are solubilized using DMSO to allow uniform color distribution. The absorbance is typically measured at 570 nm, with higher absorbance indicating greater cell viability.
Figure 1. Principle of the MTT Assay
Advantages of the MTT assay include its simplicity, low cost, and reliable results. It is commonly used in cell proliferation studies, cytotoxicity testing, and drug screening.
3. CCK-8 Assay: An Upgraded Version of the MTT Method
The Cell Counting Kit-8 (CCK-8) assay is considered an improved version of the MTT assay. It uses a more sensitive substrate, WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt). WST-8 is reduced by cellular dehydrogenases in viable cells to produce a water-soluble orange formazan product, which does not require additional solubilization steps and can be measured directly in the culture medium. Detection is performed at 450 nm.
Figure 2. Principle of the CCK-8 Assay
Compared with MTT, the CCK-8 assay offers better linearity, higher sensitivity, and lower cytotoxicity, making it more suitable for long-term culture and repeated measurement. However, it is relatively more expensive and may still be influenced by certain cell types or extreme conditions.
4. Metabolic Activity Assay: Monitoring the Cellular “Powerhouse”
ATP (adenosine triphosphate) content assays are based on the principle that viable cells continuously synthesize ATP, while ATP levels rapidly decline upon cell death. By using systems such as luciferase-luciferin, ATP content can be accurately quantified, providing a direct measure of cellular metabolic activity.
Figure 3. Principle of ATP-Based Viability Assay
This method offers insights into cellular energy metabolism and is valuable in studies involving physiological and pathological conditions. However, ATP assays are technically more complex, requiring precise handling and specialized analysis. Results may vary depending on cell type and experimental conditions, necessitating comprehensive evaluation.
5. Flow Cytometry: A Multiparametric Tool for Analyzing Viability
Flow cytometry is a powerful technique that enables high-throughput, multiparameter analysis of cells. It provides rich biological information including cell size, granularity, DNA content, cell cycle status, and viability. Cell viability is typically assessed using selective fluorescent dyes.
Common Fluorescent Dyes for Viability Detection:
Propidium Iodide (PI): Cannot penetrate intact membranes of live cells but enters dead cells, binding to nucleic acids and emitting red fluorescence.
Calcein-AM: Permeates live cell membranes and is converted by intracellular esterases into green-fluorescent calcein.
7-AAD: Similar to PI, stains dead cells with bright red fluorescence but is excluded by viable cells.
Flow cytometry offers advantages such as rapid detection, high throughput, and the ability to analyze multiple parameters simultaneously. It can analyze thousands of cells in minutes, generating comprehensive data. However, the technique requires costly equipment, skilled operation, and careful optimization for certain complex samples or cell types to ensure accurate results.
Conclusion
The above methods are among the most widely used techniques for evaluating cell viability and proliferation. Each has its own unique principles, advantages, and limitations. In real-world research or medical applications, assay selection should be based on specific objectives, cell types, and experimental conditions. Often, a combination of multiple methods provides the most reliable and comprehensive insights into cellular vitality.
Product Overview
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Product Name |
Specification |
Cat No. |
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100 T/500 T/1000 T |
40203ES60/76/80 |
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1000 T/10000 T |
40202ES80/92 |
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10 mL/100 mL/10×100 mL |
40210ES10/60/80 |