What are the different methods to monitor cell viability?

Different Methods to Monitor Cell Viability
Monitoring cell viability is a critical aspect of animal cell culture experiments. It helps in assessing the health, growth and physiological condition of cultured cells. Cell viability refers to the proportion of living cells in a population, and its measurement is important for applications like cytotoxicity testing, drug screening, vaccine production and bioprocess optimization. Viability assays work on different principles such as membrane integrity, metabolic activity, enzyme function, and dye uptake or exclusion. There are several reliable methods available to evaluate cell viability and each has its own advantages and limitations.

Here are the six most widely used and experimentally validated methods to monitor cell viability in animal cell culture:

1. Trypan Blue Exclusion Assay

This is one of the most classical and basic methods. Trypan blue is a dye that cannot enter live cells due to intact cell membranes. Only dead cells with compromised membranes allow the dye to enter and appear blue under the microscope. The live cells remain unstained. Using a hemocytometer, both live and dead cells are manually counted. Though this method is simple, quick, and inexpensive, it lacks sensitivity and is not ideal for high-throughput screening.

2. MTT, XTT, and MTS Assays (Metabolic Activity Assays)

These are colorimetric assays based on the reduction of tetrazolium salts by mitochondrial dehydrogenase enzymes present in metabolically active cells. MTT turns into purple formazan crystals, XTT and MTS into soluble formazan products. The amount of formazan formed is directly proportional to the number of viable cells and is measured using a spectrophotometer. These assays are widely used in drug screening and cytotoxicity studies.

3. Alamar Blue (Resazurin Reduction Assay)

This is a fluorometric and colorimetric method where resazurin, a blue non-toxic dye, is reduced to pink and fluorescent resorufin by viable cells. This assay is non-destructive, allows continuous monitoring, and is more sensitive than MTT. It is also suitable for long-term assays and small-scale experiments where cell preservation is important.

4. ATP-Based Luminescence Assay

This assay measures the level of intracellular ATP, which is an indicator of metabolically active and live cells. The luciferase enzyme converts ATP into light, which is then measured using a luminometer. The amount of light produced is directly proportional to the number of viable cells. This method is highly sensitive and quick but more expensive than others.

5. Annexin V and Propidium Iodide (PI) Staining by Flow Cytometry

This method allows detection of different cell death stages. Annexin V binds to phosphatidylserine, which is externalized on apoptotic cells, while PI enters only dead or late apoptotic cells. By using flow cytometry, cells can be classified as live, early apoptotic, or necrotic. This method provides very detailed results but requires advanced instrumentation and technical skill.

6. Calcein-AM and Ethidium Homodimer-1 (Live/Dead Assay)

This is a dual-fluorescence assay where live cells convert non-fluorescent Calcein-AM to green fluorescent calcein, while Ethidium Homodimer-1 stains the nuclei of dead cells red. This method is accurate, provides visual confirmation under fluorescence microscopy, and is often used in 3D cell cultures or tissue imaging.

[Note: Some researchers also include gelatin exclusion assay, LDH release assay and clonogenic assay under viability assays, but they are generally considered supportive or indirect methods.]






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