Are you uncertain which assay to use: proliferation, viability, or cytotoxicity? When starting out in cell culture, many researchers find these concepts overlapping and confusing. Yet, selecting the right one is crucial, not trivial, as it directly impacts the validity of your data. Let's break down the differences so you can confidently match the method to your experimental goal.
Table of Contents
1. Quick Guide: Key Differences Between Proliferation, Viability & Cytotoxicity
2. Overview of Cell Viability Assay Methods
3. How to Choose the Right Cell Viability Assay Method?
4. Overview of Cell Cytotoxicity Assay Methods
5. How to Choose the Right Cell Cytotoxicity Assay Method?
6. Overview of Cell Proliferation Assay Methods
01 Quick Guide: Key Differences Between Proliferation, Viability & Cytotoxicity
(1) Cell Viability: Refers to the proportion of live cells within the total cell population. The Trypan Blue exclusion assay is the classic method for determining live/dead cells, while assays like ATP/CCK-8/MTT/MTS measure viability indirectly by quantifying cellular metabolic activity[1, 2].
(2) Cell Proliferation: Refers to the ability of living cells to divide or the rate of DNA synthesis. This is typically assessed via BrdU/EdU incorporation methods[3].
(3) Cell Cytotoxicity: Measures the extent of damage or death caused to cells by external factors (e.g., drugs, chemicals, environmental stress). A standard method is the Lactate Dehydrogenase (LDH) release assay.
Table 1. Anti-cancer Drug Screening (Practical Example)
|
Cell Status |
Outcome |
|
High Toxicity, Low Viability, Low Proliferation |
Drug exhibits strong cytotoxicity (may require dose optimization or a more specific target). |
|
Low Toxicity, Normal Viability, Low Proliferation |
Drug specifically inhibits proliferation (an ideal candidate for further study). |
|
Low Toxicity, High Viability, High Proliferation |
Drug is ineffective; cancer cells continue to proliferate rapidly. |
02 Overview of Cell Viability Assay Methods
Table 2. Comparison of Cell Viability Assay Methods
|
Assay Method |
Instrument |
Core Principle |
Assay Time |
Key Features |
|
ATP Assay |
Multi-mode Microplate Reader (Chemiluminescence) |
Quantifies ATP from viable cells using the luciferin-luciferase system. Signal intensity is linearly proportional to live cell number. |
10-20 min |
Fast, highly sensitive. Requires a dedicated luminescence detector. |
|
CCK-8 Assay |
Microplate Reader |
WST-8 is reduced by cellular dehydrogenases to a water-soluble formazan. Absorbance correlates with viable cell number. |
2-5 h |
Simple, no solubilization step, low cytotoxicity. |
|
MTT Assay |
Microplate Reader |
MTT is reduced by mitochondrial enzymes to an insoluble purple formazan crystal. Absorbance correlates with viable cell number. |
2-5 h |
High sensitivity, but requires a solubilization step. |
|
Flow Cytometer/Fluorescence Microscope |
Calcein-AM stains live cells (green), PI stains dead cells (red). |
40-60 min |
Directly distinguishes live/dead cells; suitable for multiparameter analysis. |
|
|
Trypan Blue Staining |
Microscope |
Dye is excluded by live cells but stains dead cells blue. |
10-15 min |
Low throughput; accuracy depends on manual counting. |
03 How to Choose the Right Cell Viability Assay Method?
If you have a chemiluminescence reader and need high speed/sensitivity:
→ Cell Viability Chemiluminescence Assay Kit (E-BC-F200)
A quantitative ATP method that is precise and fast.
Principle: Utilizes the specific enzymatic reaction between luciferase, ATP, and luciferin. The luminescence intensity is strictly linear with ATP concentration, enabling accurate quantification of viable cells.
If you have a standard plate reader and want a cost-effective, simple option:
→ Enhanced Cell Counting Kit 8 (WST-8/CCK8) (E-CK-A362)
A classic, stable, and user-friendly colorimetric choice.
Principle: WST-8 is reduced by mitochondrial dehydrogenases to a water-soluble formazan dye. The amount of formazan generated is proportional to the number of viable cells, which is measured by absorbance at 450 nm.
Table 3. Cell Viability Assay Product List
|
Cat. No. |
Product Name |
Size |
Instrument |
Application |
|
E-BC-F200 |
Cell Viability Chemiluminescence Assay Kit |
48 T/96 T |
Chemiluminescence/Multi-mode Microplate Reader |
Cell Viability |
|
E-CK-A362 |
Enhanced Cell Counting Kit 8 (WST-8/CCK8) |
100 Tests/500 Tests |
Microplate Reader |
Cell Viability / Proliferation / Cytotoxicity |
04 Overview of Cell Cytotoxicity Assay Methods
Table 4. Comparison of Cell Cytotoxicity Assay Methods
|
Assay Method |
Instrument |
Core Principle |
Assay Time |
Key Features |
|
LDH Release Assay |
Microplate Reader |
Measures LDH enzyme released from damaged cells into the culture medium via a colorimetric reaction. |
60-90 min |
Straightforward; may be interfered with by serum components. |
|
Luciferase Reporter Gene Assay |
Chemiluminescence/Multi-mode Microplate Reader |
Quantifies the release of a luciferase reporter enzyme upon cell lysis. |
25 min |
Highly sensitive, but requires transfected cells with a reporter construct. |
05 How to Choose the Right Cell Cytotoxicity Assay Method?
Looking for a rapid, universal method for high-throughput screening?
→ Lactate Dehydrogenase (LDH) Cytotoxicity Colorimetric Assay Kit (E-BC-K771-M)
Requires only supernatant detection; instrument-friendly and efficient.
Principle: LDH catalyzes a reaction generating NADH, which reduces WST-8 to a yellow formazan. The absorbance at 450 nm is proportional to LDH activity and, thus, cytotoxicity.
Table 5. Cell Cytotoxicity Product List
|
Cat. No. |
Product Name |
Size |
Instrument |
Application |
|
Lactate Dehydrogenase (LDH) Cytotoxicity Colorimetric Assay Kit |
96 T |
Microplate Reader |
Cell Cytotoxicity |
|
|
Dual Luciferase Reporter Gene Luminescence Assay Kit (Glow Type) |
48 T/96 T |
Multi-mode Microplate Reader |
Cell Cytotoxicity, Molecular Experiments |
06 Overview of Cell Proliferation Assay Methods
Table 6. Comparison of Cell Proliferation Assay Methods
|
Assay Method |
Instrument |
Core Principle |
Assay Time |
Key Features |
|
MTS Assay |
Microplate Reader |
MTS is reduced by cellular dehydrogenases to a soluble formazan. Absorbance correlates with metabolic activity and cell number. |
3-7 h |
Simpler than MTT (no solubilization), but slightly less sensitive than CCK-8. |
|
BrdU Assay |
Flow Cytometer / Microplate Reader |
Detects BrdU incorporated into newly synthesized DNA using specific antibodies. |
12-36 h |
Direct measure of DNA synthesis. Requires DNA denaturation for antibody access. |
|
EdU Assay |
Flow Cytometer / Fluorescence Microscope / Microplate Reader |
Uses a click chemistry reaction to detect EdU incorporated into new DNA. |
5-18 h |
Simpler than BrdU (no denaturation). Flexible for various readouts. |
|
DNA Content Analysis (PI) |
Flow Cytometer |
PI stains DNA. The fluorescence intensity and cell cycle distribution (G1/G0, S, G2/M) indicate proliferation status. |
2-3 h |
Cannot distinguish between quiescent and actively cycling cells in G0/G1 phase alone. |
References:
[1] Stoddart, Martin J. Mammalian cell viability : methods and protocols[M]. 2011.
[2] Gilbert D F, Friedrich O. ell Viability Assays[J]. Methods in Molecular Biology, 2017. DOI:10.1007/978-1-4939-6960-9.
[3] Ma Y, Liu E, Fan H, et al. RBM47 promotes cell proliferation and immune evasion by upregulating PDIA6: a novel mechanism of pancreatic cancer progression[J]. Journal of Translational Medicine, 2024, 22(1). DOI:10.1186/s12967-024-05970-6.
