CellTiter-Glo® Luminescent Cell Viability Assay
Quick Facts
| Feature | Information |
|---|---|
| Assay Name | CellTiter-Glo® Luminescent Cell Viability Assay |
| Assay Type | Cell Viability Assay |
| Detection Method | Luminescence |
| Principle | ATP Quantification Using Luciferase |
| Measured Parameter | Intracellular ATP |
| Readout | Relative Luminescence Units (RLU) |
| Suitable Cell Types | Adherent and Suspension Cells |
| Typical Assay Time | 20-30 minutes |
| Equipment Required | Luminescence Plate Reader or Luminometer |
Overview
The CellTiter-Glo® Luminescent Cell Viability Assay is a homogeneous, ATP-based assay used to determine the number of metabolically active cells in culture.
Unlike colorimetric assays such as MTT or MTS, CellTiter-Glo measures intracellular ATP, which rapidly declines when cells lose viability. Because ATP is present only in metabolically active cells, the luminescent signal generated by the assay is directly proportional to the number of viable cells.
The assay combines cell lysis and ATP detection into a single step, making it rapid, highly sensitive, and well suited for high-throughput screening (HTS).
Scientific Principle
ATP serves as the universal energy currency of living cells. Viable cells continuously synthesize ATP through glycolysis and oxidative phosphorylation. Following cell death, ATP production ceases and intracellular ATP is rapidly degraded.
The CellTiter-Glo reagent contains a proprietary cell lysis buffer together with firefly luciferase and its substrate, luciferin. Upon addition, cells are lysed, ATP is released, and the luciferase reaction generates light.
The amount of light produced is directly proportional to the ATP concentration and therefore to the number of viable cells.
Reaction
Luciferin + ATP + O₂
↓ Luciferase
Oxyluciferin + AMP + PPi + Light
Higher ATP concentrations produce stronger luminescent signals.
Applications
The CellTiter-Glo assay is widely used in:
- Cell viability studies
- Drug discovery
- High-throughput compound screening
- Cytotoxicity testing
- Cancer biology
- Stem cell research
- Toxicology
- Organoid research
- 3D spheroid viability studies
Suitable Cell Types
The assay is compatible with:
- Adherent cell lines
- Suspension cell lines
- Primary cells
- Stem cells
- Patient-derived cells
- Organoids
- Tumor spheroids
Materials Required
- CellTiter-Glo® Reagent
- White-walled microplates (recommended)
- Cell culture medium
- Micropipettes and sterile tips
- Orbital shaker (optional)
- Luminescence plate reader
General Procedure
1. Seed Cells
Seed cells at the desired density and allow sufficient time for attachment or recovery before treatment.
2. Perform Experimental Treatment
Treat cells according to the experimental design.
Include untreated, vehicle, blank and positive control wells.
3. Equilibrate Reagents
Allow both the CellTiter-Glo reagent and the assay plate to equilibrate to room temperature before beginning the assay to improve reaction consistency.
4. Add CellTiter-Glo Reagent
Add an equal volume of CellTiter-Glo reagent directly to each well. The reagent simultaneously lyses the cells and initiates the ATP-dependent luciferase reaction.
Note: White assay plates are recommended because they maximize light reflection and improve luminescence sensitivity.
5. Mix
Mix the plate gently for approximately 2 minutes to ensure complete cell lysis.
Avoid vigorous shaking, which may introduce bubbles and interfere with luminescence measurements.
6. Incubate
Incubate the plate for approximately 10 minutes at room temperature to allow the luminescent signal to stabilize.
7. Measure Luminescence
Measure luminescence using a compatible plate reader or luminometer.
Higher luminescence corresponds to higher intracellular ATP levels and therefore greater numbers of viable cells.
Recommended Controls
| Control | Purpose |
|---|---|
| Blank | Measures background luminescence |
| Untreated Cells | Represents 100% viability |
| Vehicle Control | Determines solvent effects |
| Positive Cytotoxicity Control | Confirms assay performance |
Data Interpretation
| Observation | Interpretation |
|---|---|
| High luminescence | High ATP concentration and high cell viability |
| Moderate luminescence | Reduced viability or slower proliferation |
| Low luminescence | Significant cell death or metabolic inhibition |
| Background signal | Absence of viable cells |
Advantages
- Rapid one-step assay
- Highly sensitive
- Excellent reproducibility
- No washing steps required
- Wide dynamic range
- Suitable for high-throughput screening
- Compatible with automated liquid handling
- Applicable to both 2D and 3D cultures
Limitations
- Measures ATP rather than absolute cell number.
- ATP levels may change independently of cell number due to metabolic alterations.
- Cell lysis prevents downstream analysis using the same sample.
- Requires a luminescence plate reader.
Comparison with Other Cell Viability Assays
| Feature | CellTiter-Glo | MTT | Resazurin |
|---|---|---|---|
| Detection | Luminescence | Colorimetric | Fluorescence/Colorimetric |
| Measures | ATP | Mitochondrial activity | Cellular metabolism |
| Cell Lysis | Yes | No | No |
| Washing Required | No | Solubilization step required | No |
| High-Throughput Compatible | Excellent | Moderate | Excellent |
| Sensitivity | Very High | Moderate | High |
Troubleshooting
| Observation | Possible Cause | Corrective Action |
|---|---|---|
| Weak luminescence | Low ATP levels or insufficient cells | Verify cell density and cell health |
| High background | Contaminated reagent or poor blank correction | Include fresh blank wells |
| Variable results | Uneven cell seeding | Seed cells uniformly |
| Unexpectedly low signal | Drug-induced metabolic inhibition | Confirm results using complementary assays |
Common Beginner Mistakes
- Using black plates instead of white plates.
- Reading the plate immediately after reagent addition.
- Creating air bubbles while mixing.
- Comparing ATP levels between metabolically different cell types.
- Assuming ATP always equals cell number under all experimental conditions.
Quality Control
Before beginning the assay, verify:
- Proper reagent storage.
- Uniform cell seeding.
- Appropriate controls.
- Correct plate reader settings.
- Fresh reagent preparation.
Frequently Asked Questions
Why does CellTiter-Glo measure ATP?
ATP is rapidly lost following cell death, making it an excellent indicator of metabolically active, viable cells.
Why are white plates recommended?
White plates reflect emitted light, producing stronger and more reproducible luminescent signals than clear or black plates.
Can CellTiter-Glo distinguish apoptosis from necrosis?
No. The assay measures total ATP and therefore overall cell viability. Additional assays such as Annexin V, Caspase assays, or TUNEL assays are required to determine the mechanism of cell death.
Can CellTiter-Glo be used for organoids?
Yes. The assay is widely used for organoids and tumor spheroids, although incubation times may need optimization for larger 3D structures.
Key Takeaways
- CellTiter-Glo is a luminescent ATP-based viability assay.
- ATP concentration is proportional to the number of metabolically active cells.
- The assay combines cell lysis and ATP detection into a single step.
- It is one of the most sensitive assays for high-throughput screening.
- White plates provide optimal luminescence detection.
- Appropriate controls are essential for accurate data interpretation.
- Complementary assays should be used to identify specific mechanisms of cell death.