“Is my cell still alive?” is one of the most common questions in cell biology, and there are at least a dozen ways to answer it. The right cell viability assay depends on what you’re measuring (metabolic activity, membrane integrity, ATP), how many samples you have, and how sensitive you need to be.
What “cell viability” actually means
No assay measures life directly. Each measures a proxy:
- Metabolic activity (MTT, MTS, XTT, alamarBlue)
- ATP content (CellTiter-Glo)
- Membrane integrity (trypan blue, LDH release)
- DNA content (CyQUANT, Hoechst)
These can disagree, especially for stressed cells.
The metabolic dye family
MTT
The original. Live cells reduce yellow MTT to purple formazan crystals via mitochondrial dehydrogenases. The crystals are insoluble and require a solubilization step (DMSO or detergent) before reading at 570 nm. Cheap, well-established, but the extra step adds variability and the formazan is toxic, making it endpoint-only.
MTS
An MTT improvement. The formazan is water-soluble — read directly. Faster, more reproducible, same chemistry. CellTiter 96 AQueous is the common commercial version.
alamarBlue (resazurin)
Live cells reduce blue, non-fluorescent resazurin to pink, fluorescent resorufin. Read by absorbance or fluorescence (excitation 560 nm, emission 590 nm). Non-toxic at standard concentrations, so you can monitor the same plate over multiple time points. Excellent for kinetic studies.
The ATP-based assay
CellTiter-Glo
A luciferase-based luminescent assay. Cells are lysed, ATP reacts with luciferin and luciferase to produce light. Extremely sensitive (detects as few as 10 cells per well), wide dynamic range, fast (10 minutes), and very reproducible. Downsides: endpoint assay (cells are killed), and significantly more expensive per well than dye-based options.
Side-by-side comparison
| Assay | Detection | Time | Sensitivity | Live-cell? | Cost |
|---|---|---|---|---|---|
| MTT | Absorbance | 4 h + solubilize | Moderate | No | Low |
| MTS | Absorbance | 1–4 h | Moderate | No | Low |
| alamarBlue | Fluorescence/Abs | 1–4 h | High | Yes | Medium |
| CellTiter-Glo | Luminescence | 10 min | Very high | No | High |
| Trypan blue | Visual | 5 min | Low | No | Very low |
How to choose
- Drug screening at scale: CellTiter-Glo for sensitivity, or MTS for budget.
- Kinetic studies: alamarBlue, since you can read the same plate repeatedly.
- Quick check at trypsin/seeding: Trypan blue with a hemocytometer or automated counter.
- Co-cultures or 3D cultures: ATP-based or fluorescent assays generally outperform absorbance.
Common mistakes
- Assuming linearity at extreme cell densities — always run a standard curve
- Reading too early or too late — follow incubation guidelines
- Ignoring background controls — cell-free wells with same media and reagents are essential
- Over-interpreting small differences — calculate IC50 with proper curve fitting
Different assays sometimes give different answers because they measure different things. For high-stakes conclusions, run two orthogonal viability assays — agreement gives a much stronger result than either alone.
