Six Common Methods for Cell Viability Assay: Principles, Applications and ANTBIO Premium Solutions

In the vast field of biomedical research, cell viability assay plays a pivotal role. It not only helps scientists evaluate the survival status of cells but also gain in-depth insights into cellular responses to drugs, environmental changes, or pathological processes. Typically, cell viability assay involves measuring cell proliferation, cytotoxicity, and cell death. Today, we introduce six common methods for cell viability assay: ATP Bioluminescence Assay, LDH Assay, MTT Colorimetric Assay, CCK-8 Assay, Fluorescent Dye Assay, and Apoptosis Assay, to unravel the scientific mysteries of cell vitality together.

1. Core Principles of Six Common Cell Viability Assay Methods

1.1 ATP Bioluminescence Assay

The intracellular content of ATP (Adenosine Triphosphate) is a direct indicator of cellular energy metabolism. The ATP Bioluminescence Assay evaluates cell viability by measuring intracellular ATP levels. This method boasts high sensitivity and a wide dynamic range, making it particularly suitable for experiments requiring high-sensitivity detection.

The main principle is that under aerobic conditions, luciferin reacts with ATP in the presence of luciferase and Mg²⁺ to form a luciferin-AMP complex, releasing pyrophosphate (PPi). Subsequently, the luciferin-AMP complex further reacts with O₂ to generate oxyluciferin, along with the release of CO₂ and H₂O. During this process, the excited oxyluciferin emits photons when returning to the ground state. The number of photons is proportional to the ATP content, so the ATP level can be quantified by measuring the light intensity.

1.2 LDH Assay

Lactate Dehydrogenase (LDH) is an enzyme widely present in cells, playing a key role in the glycolytic pathway by catalyzing the reversible reaction between lactate and pyruvate. LDH catalyzes lactate to produce pyruvate, which reacts with 2,4-dinitrophenylhydrazine to form pyruvate dinitrophenylhydrazone, which appears reddish-brown in an alkaline solution. The depth of the color is proportional to the concentration of pyruvate, and the LDH activity can be calculated by measuring the OD value.

In cell viability assay, LDH release is usually used as an indicator of cell membrane integrity. When the cell membrane is damaged, intracellular LDH is released into the culture medium. By determining the LDH activity in the culture medium, the degree of cell damage or death can be evaluated.

1.3 MTT Colorimetric Assay

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Colorimetric Assay is a classic method for cell viability detection. It indirectly reflects cellular metabolic activity by measuring the activity of dehydrogenases in living cells, which reduce MTT to purple formazan products. This method is simple to operate and low-cost, making it a commonly used screening tool in laboratories.

1.4 CCK-8 Assay

The CCK-8 (Cell Counting Kit-8) Assay is a colorimetric method based on WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt). It evaluates cell viability by measuring the activity of mitochondrial dehydrogenases in cells. The CCK-8 Assay has high sensitivity and a wide dynamic range, making it suitable for viability detection of various cell types.

1.5 Fluorescent Dye Assay

The Fluorescent Dye Assay uses specific fluorescent dyes, such as Calcein-AM or CFSE, to label living cells. These dyes are metabolized into fluorescent products in living cells and can be detected by flow cytometry or fluorescence microscopy. The Fluorescent Dye Assay can provide intuitive images of cell viability and is suitable for cell proliferation and migration research.

1.6 Apoptosis Assay

Apoptosis is a process of programmed cell death closely related to cell viability. By staining with Annexin V and PI (Propidium Iodide), the externalization of phosphatidylserine on the cell surface can be detected, thereby evaluating the degree of apoptosis. This method is of great significance for studying cell death mechanisms and drug toxicity.

2. Comparison of Advantages and Disadvantages of Six Common Methods

Each method has its specific application scenarios and limitations. The selection of an appropriate assay method needs to be determined based on the specific needs and conditions of the experiment.

3. In-Depth Introduction to ATP Bioluminescence Assay (High Sensitivity & Rapid Detection)

Among the six common methods, the ATP Bioluminescence Assay stands out due to its high sensitivity, simple operation, and rapid detection. Below is a detailed introduction to the usage method of the ATP Assay Kit (Catalog No.: abs580117) launched by ANTBIO's sub-brand Absin.

3.1 Product Composition

3.2 Usage Method

3.2.1 Reagents and Equipment to Be Prepared

•         Phosphate Buffered Saline (PBS) pH7.4 for cell culture

•         Triton X-100

•         Adjustable volume pipettes and tips

•         Centrifuge tubes and white 96-well plates

•         Chemiluminescence microplate reader

3.2.2 Pre-Experimental Preparation

3.2.2.1 Sample Preparation

•         Preparation of cell culture supernatant: Seed the cells to be tested into culture plates, treat with interfering factors, then directly aspirate the cell supernatant. For suspension cells, centrifuge at 300g for 5min at 4°C and collect the supernatant.

•         Preparation of cell lysate supernatant: Collect the cells to be tested (1×10⁶-1×10⁷) into a 5mL centrifuge tube, centrifuge at 300g for 5min at 4°C, discard the supernatant. Add 200μL PBS containing 0.1% Triton X-100, lyse on ice for 30min, then centrifuge at 10000g for 10min at 4°C and collect the supernatant.

•         Preparation of tissue lysate supernatant: Collect the tissue to be tested (20-50mg) into a glass homogenizer or automatic homogenization tube, add 500μL PBS containing 1% Triton X-100, homogenize for 1min, centrifuge at 10000g for 10min at 4°C and collect the supernatant.

Note: If samples are not tested immediately, store them at -80°C. Before formal determination, appropriately dilute the samples with ATP Assay Buffer according to pre-experimental results.

3.2.2.2 Preparation of Standards

In a 1.5mL centrifuge tube, add 990μL ATP Assay Buffer, then add 10μL of 100μM ATP Standard to prepare 1μM ATP Standard. Take another 8 1.5mL centrifuge tubes, add 200μL ATP Assay Buffer to each, then sequentially add 200μL of 1μM ATP Standard for serial two-fold dilution to obtain concentrations of 0.5, 0.25, 0.125, 0.0625, 0.0312, 0.0156, 0.0078, and 0.0039μM.

3.2.2.3 Preparation of Detection Working Solution

Prepare an appropriate amount of ATP detection working solution according to the number of samples to be tested by mixing the reagents in the following proportions. The mixed reagents serve as the ATP detection working solution.

3.2.3 Operation Steps

1.       Set up blank control wells, standard wells, and sample wells in a white 96-well plate. Add 50μL ATP Assay Buffer to blank control wells, 50μL gradient concentration ATP Standard (0.0039-1μM) to standard wells, and 50μL samples to sample wells, as shown in the following table:

2.       Add 50μL of ATP detection working solution to each well as shown in the table, mix well, and incubate at room temperature for 5min.

3.       After the reaction is completed, measure the Relative Luminescence Units (RLU) using a chemiluminescence microplate reader.

4.       Result Calculation: Plot a standard curve with the ATP standard concentration as the abscissa and the RLU value as the ordinate, and obtain the functional relationship between the abscissa and ordinate. Then calculate the ATP concentration in each sample using the RLU value of the sample.

4. ANTBIO Recommended Products for Cell Viability Assay

To meet the diverse needs of cell viability research, ANTBIO's sub-brand Absin has launched a series of high-quality detection kits and reagents, covering all six common cell viability assay methods. The detailed product information is as follows:

5. ANTBIO Recommended Cell Staining Products

In addition to cell viability assay products, ANTBIO also provides a full range of cell staining products to support in-depth cellular research. These products cover multiple application scenarios such as cell membrane staining, nuclear staining, organelle staining, and ion detection, with excellent performance and high reliability.

6. The Significance of Cell Viability Assay in Biomedical Research

Cell viability assay is a bridge connecting basic research and clinical applications. Through the above six common methods, researchers can more accurately evaluate the survival status of cells, providing important information for disease treatment and drug development. With the continuous advancement of biotechnology, more innovative detection methods may emerge in the future, revealing more mysteries of cell vitality for us.

7. About AN BIO PTE. LTD.

AN BIO PTE. LTD. is committed to providing high-quality, reliable biological research reagents and comprehensive solutions for global scientific researchers. With its professional sub-brand Absin, we focus on the research and development and production of cell biology, immunology, and molecular biology-related products. Our products are widely used in academic research, drug development, and clinical diagnosis, winning the trust and recognition of numerous researchers.

We adhere to the core values of "quality first, customer-oriented", continuously optimize product performance and service quality, and strive to become a trusted partner in the field of life sciences, contributing to the progress of global biomedical research.

8. Disclaimer

This article is compiled and interpreted by AI based on the original work with DOI: 10.1002/advs.202413562. All intellectual property rights (such as images, data) of the original publication belong to the journal and the research team. For any infringement, please contact us immediately and we will take prompt action.

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