Cell viability assays are essential techniques used to assess the health and survival of cells in various experimental conditions. These assays help determine how treatments, drugs, or environmental conditions affect cell growth and function. Here's a detailed technical overview of cell viability assay kits:

Types of Cell Viability Assays

1. Colorimetric Assays:
   • MTT Assay:
     • Principle: Measures cell viability based on the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan crystals by mitochondrial enzymes in live cells.
     • Procedure: Cells are incubated with MTT, and the resulting formazan crystals are solubilized and quantified by measuring absorbance at 570 nm using a spectrophotometer.
   • XTT Assay:
     • Principle: Involves the reduction of XTT (sodium 3'-[1-(phenylamino)-carbonyl]-3,4-tetrazolium]-bis (4-methoxy-6-nitro) phenyl) to a soluble formazan dye.
     • Procedure: Cells are incubated with XTT, and the absorbance of the resulting soluble formazan dye is measured at 450 nm or 490 nm.
2. Fluorometric Assays:
   • Resazurin Assay:
     • Principle: Uses resazurin, a blue non-fluorescent dye, which is reduced to resofurin, a pink fluorescent dye, by viable cells.
     • Procedure: Cells are incubated with resazurin, and the fluorescence of resofurin is measured at an emission wavelength (e.g., 585 nm) after excitation (e.g., 560 nm).
   • Calcein AM Assay:
     • Principle: Calcein AM (acetoxymethyl ester) is converted to calcein, a green fluorescent dye, by intracellular esterases in viable cells.
     • Procedure: Cells are incubated with Calcein AM, and fluorescence is measured using a fluorometer or fluorescence microscope.
3. Luminescent Assays:
   • ATP Assay:
     • Principle: Measures cellular ATP levels, which correlate with viable cell numbers, using a luciferase-based reaction.
     • Procedure: Cells are lysed, and ATP is quantified by the light emitted in the reaction with luciferase. Light intensity is measured using a luminometer.
   • CellTiter-Glo® Assay:
     • Principle: Uses a luminescent signal generated from the reaction of ATP with a luciferase enzyme to measure viable cell numbers.
     • Procedure: Cells are lysed, and the luminescence is measured directly to determine ATP levels and thus cell viability.

Key Reagents and Components

• Dyes and Substrates: Include MTT, XTT, WST-1, resazurin, Calcein AM, Propidium Iodide, and others, depending on the assay type.
• Enzymes: Includes luciferase for ATP assays or other enzymatic components used in the detection process.
• Buffers and Reagents: Solutions for cell lysis, dye solubilization, or other assay-specific preparations.
• Standards and Controls: Used for calibration and to ensure the accuracy of the assay.

Procedure

• Cell Preparation: Seed cells in appropriate culture plates or wells. Allow them to adhere and grow to the desired confluence.
• Treatment: Apply experimental treatments or drugs as needed and incubate for the specified duration.
• Assay Execution:
  • Colorimetric Assays: Add the assay reagent (e.g., MTT or XTT) to cells, incubate, and measure absorbance.
  • Fluorometric Assays: Add fluorogenic dyes (e.g., resazurin or Calcein AM), incubate, and measure fluorescence.
  • Luminescent Assays: Lyse cells, add reagents for luminescent detection (e.g., CellTiter-Glo®), and measure light emission.
  • Dye Exclusion Assays: Mix cells with dye, count using a microscope or automated counter, and calculate the percentage of viable cells.
• Data Analysis: Analyze the results to determine cell viability. Compare treated samples with control or untreated samples to assess the effects of treatments.

Calibration and Validation

• Calibration: Use known cell concentrations or control samples to establish calibration curves for accurate measurement of cell viability.
• Validation: Confirm assay performance by evaluating parameters such as sensitivity, specificity, accuracy, and reproducibility. Validate results with standard samples or through comparison with other viability assays.

Applications

• Drug Screening: Evaluate the cytotoxicity or efficacy of new drugs or compounds.
• Cancer Research: Assess the effects of therapies on cancer cell lines.
• Cell Biology: Study cell proliferation, toxicity, and general cell health.
• Toxicology: Test the impact of environmental or chemical agents on cell viability.