Cell proliferation lies at the core of growth, development, and tissue renewal. Likewise, cytotoxicity—the ability of a compound or condition to damage or kill cells—is a fundamental readout across drug screening, toxicology, and basic research. Together, these measurements form the backbone of modern biomedical research.

Depending on your lab setup, you may run these assays using

a multimode plate reader (chemiluminescence, absorbance, fluorescence),

a fluorescence microscope, or a flow cytometer.

Below, we break down the most commonly used assays, how they work, how to interpret results, and how to choose the right method for your experiment.

1. Overview of Common Cell Proliferation & Cytotoxicity Assays

Table 1. Quick Selection of Chemiluminescence-Based Cell Proliferation & Cytotoxicity Assays

Category	Assay / Method	Detection Principle	Detection Instrument / Notes
Chemiluminescence / Colorimetric Assays	MTT	Cell metabolism-based detection	Microplate reader
	CCK-8	Chemiluminescence / colorimetric assay	Microplate reader
	ATP assay	Cell viability via ATP quantification	Multifunctional microplate reader
	LDH assay	Cell cytotoxicity / membrane integrity	Microplate reader
	Total protein assay	Cell growth / proliferation	Microplate reader
Fluorescent Dye-Based Assays	BrdU	DNA synthesis detection	Fluorescence microscope / Flow cytometer
	EdU	DNA synthesis detection	Flow cytometer / Fluorescence microscope
	Live/dead staining	Viability assay	Fluorescence microscope / Flow cytometer
	Fluorescent cell tracking dyes	Live cell tracing	Fluorescence microscope / Cell imaging systems

2. Key Principles of Fluorescence-Based Cell Proliferation & Cytotoxicity Assays

DNA Synthesis Detection

Fluorescent nucleoside analogs, such as BrdU (Bromodeoxyuridine) and EdU (5-Ethynyl-2’-deoxyuridine), are incorporated into newly synthesized DNA during cell proliferation. Detection is performed via:

- BrdU: Fluorescent antibody staining followed by flow cytometry or fluorescence microscopy.
- EdU: Click chemistry-based fluorescent labeling, compatible with high-throughput imaging or flow cytometry.

Figure 1. EDU staining principle diagram

Viability and Live/Dead Staining

Fluorescent dyes such as Calcein-AM, Propidium Iodide (PI), and Hoechst 33342 allow rapid discrimination between live and dead cells. These dyes are widely used in cytotoxicity studies, drug screening, and cell tracking experiments.

Figure 2. Cell staining results.

Cell type: A375 melanoma cells (note: A375 is a human melanoma cell line, not glioma; please confirm if "glioma" was intended).

Staining conditions: Calcein-AM (2 μM) and propidium iodide (PI, 4.5 μM) for 15 minutes.

(A) Calcein-AM channel (live cells, green fluorescence);

(B) PI channel (dead cells, red fluorescence);

Cell Tracking and Long-Term Proliferation Monitoring

Fluorescent cell tracers, including CFSE or CellTrace™ dyes, label living cells and allow monitoring of cell divisions over time using flow cytometry or live-cell imaging.

Table 2. Quick Selection of Fluorescence-Based Cell Proliferation & Cytotoxicity Assays

Method	Detection Platform	Cytotoxicity Detection	Ease of Use	Sensitivity	Assay Time	Typical Applications / Cell Types
BrdU	Fluorescence microscopy / Flow cytometry	Low	Moderate	High	2–4 h	Adherent or suspension cells; DNA synthesis analysis; drug screening
EdU	Fluorescence microscopy / Flow cytometry	Low	Easy	Very High	30–60 min	Rapid DNA synthesis detection; high-throughput assays
Live/Dead Staining (Calcein-AM / PI / Hoechst)	Fluorescence microscopy / Flow cytometry	High	Easy	High	10–60 min	Viability/cytotoxicity assays; apoptosis/necrosis studies
CellTrace™ / CFSE	Fluorescence microscopy / Flow cytometry	Low	Moderate	High	Hours–Days	Cell proliferation tracking; long-term division studies; immunology assays

3. Yeasen Cell Proliferation Assays — Fluorescent Dye Method

Product Selection Guide

Fluorescent dye-based assays provide sensitive and versatile options for monitoring cell proliferation, tracking live cells, and analyzing cell viability. Depending on your application, target organelle, and detection instrument, you can choose the optimal reagent from the following options:

Cat. No.	Product Name	Ex/Em (nm)	Staining Target	Applications
40757ES	DiR Iodide (DiIC18(7)) Cell Membrane Deep Red Fluorescent Probe	748/780	Cell membrane, deep red fluorescence	Live cell staining, in vivo imaging or tracking, exosome labeling
40725ES	DiO (DiOC18(3)) Cell Membrane Green Fluorescent Probe	484/501	Cell membrane, green fluorescence	Live cell staining, in vivo imaging
40758ES	DiD Perchlorate (DiIC18(5)) Cell Membrane Red Fluorescent Probe	644/663	Cell membrane, red fluorescence	Exosome labeling, live cell staining, staining cells/tissues with other fluorescence
40714ES	CFDA SE Cell Proliferation and Cell Tracking Kit	494/521	Cell membrane, green fluorescence	Cell proliferation colony analysis, in vivo proliferation studies
40721ES	CMFDA Live Cell Tracking Dye (Green)	492/517	Cytoplasm, green fluorescence	Cell proliferation, cell fusion, cell adhesion, MDR protein studies, pathogen labeling
40792ES	CM-DiI Live Cell Tracking Dye (Red)	548/563	Cytoplasm, red fluorescence	Live animal cell staining, general cell labeling
40747ES	Calcein-AM/PI Double Stain Kit	490/515	Calcein-AM: live cell cytoplasm, green fluorescence; PI: dead cell nucleus, red fluorescence	Live/dead cell staining
40711ES	PI (Propidium Iodide)	535/617	DNA in nucleus	Dead cell staining
40745ES	7-AAD Viability Staining Solution	546/647	DNA in nucleus	Necrotic or late-apoptotic cell staining
40202ES	Alamar Blue	560/590	Non-fluorescent in oxidized state; reduced to pink/red fluorescent product in proliferating cells	Proliferation detection in animal, fungal, and bacterial cells

Cat. No.	Product Name	Description / Fluorescence	Package / Size
40275ES60/76	YSfluor 488 EdU Imaging Kit	Cell imaging kit, green fluorescence	100/500 T
40276ES60/76/80	YSfluor 594 EdU Imaging Kit	Cell imaging kit, red fluorescence	100/500 T
40277ES60/76/80	YSfluor 647 EdU Imaging Kit	Cell imaging kit, far-red fluorescence	100/500 T
40278ES25/50	YSfluor 488 EdU Flow Cytometry Assay Kit	Cell flow cytometry kit, green fluorescence	20/50 T
40279ES25/50	YSfluor 594 EdU Flow Cytometry Assay Kit	Cell flow cytometry kit, red fluorescence	20/50 T
40280ES25/50	YSfluor 647 EdU Flow Cytometry Assay Kit	Cell flow cytometry kit, far-red fluorescence	20/50 T

บทความถัดไป

The Complete Guide to Cell Proliferation & Cytotoxicity Assays (Part II): Fluorescence-Based Methods

1. Overview of Common Cell Proliferation & Cytotoxicity Assays

2. Key Principles of Fluorescence-Based Cell Proliferation & Cytotoxicity Assays

DNA Synthesis Detection

Viability and Live/Dead Staining

Cell Tracking and Long-Term Proliferation Monitoring

3. Yeasen Cell Proliferation Assays — Fluorescent Dye Method

Product Selection Guide

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