Technical Specification: Cell Proliferation Assay Kits
1. Product Range
Solarbio cell proliferation assays utilize tetrazolium salt reduction as a proxy for cellular metabolic activity. Viable cells with active NAD(P)H-dependent oxidoreductase enzymes reduce these salts to colored formazan products. The absorbance of the formazan product is directly proportional to viable cell number.
| Product |
SKU |
Detection Principle |
Wavelength |
Assays |
| CCK-8 Cell Counting Kit |
CA1210 |
WST-8 tetrazolium reduction |
450 nm (ref 630 nm) |
500 assays (10 mL) |
| MTT Cell Proliferation Assay Kit |
CA1240 |
MTT reduction to formazan |
570 nm (ref 630 nm) |
500 assays |
| BrdU Cell Proliferation ELISA Kit |
CA1260 |
BrdU incorporation, anti-BrdU-HRP detection |
450 nm |
200 assays |
2. Product Specifications
CCK-8 Kit (CA1210)
Principle: CCK-8 contains WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] and an electron mediator (1-methoxy PMS). Dehydrogenases in viable cells reduce NAD⁺ to NADH. NADH then reduces 1-methoxy PMS, which in turn reduces WST-8 to a water-soluble orange formazan product. Unlike MTT, the WST-8 formazan is water-soluble, eliminating the need for solubilization steps.
The reaction chain:
NAD(P)H + 1-methoxy PMS (oxidized) → NAD(P)⁺ + 1-methoxy PMS (reduced)
↓
WST-8 (tetrazolium) + 1-methoxy PMS (reduced) → WST-8 formazan (orange, λmax 450 nm)
| Parameter |
Specification |
| Cell number per well |
1×10³–1×10⁵ cells (96-well) |
| CCK-8 volume |
10 μL per 100 μL culture medium |
| Incubation time |
1–4 h at 37°C |
| Detection wavelength |
450 nm (reference 630 nm) |
| Linear detection range |
500–50,000 cells/well |
| Sensitivity |
Detect ≥100 cells difference |
| Inter-assay CV |
<8% |
| Intra-assay CV |
<5% |
| Water solubility |
Complete; no solubilization step needed |
| Cytotoxicity of reagent |
Low (<5% at 4 h exposure) |
| Z′-factor (quality metric) |
>0.7 |
| Storage (dark, 2–8°C) |
12 months |
MTT Cell Proliferation Assay Kit (CA1240)
Principle: MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is a yellow tetrazolium salt that is reduced by NAD(P)H-dependent mitochondrial dehydrogenases (primarily succinate dehydrogenase) in viable cells to purple insoluble formazan crystals. The formazan must be solubilized with DMSO or acidified isopropanol before absorbance measurement.
The reaction:
MTT (yellow, λmax 440 nm) + NADH/H⁺ → MTT formazan (purple, λmax 570 nm)
(mitochondrial dehydrogenases) (insoluble in water)
| Parameter |
Specification |
| Cell number per well |
2×10³–1×10⁵ cells (96-well) |
| MTT solution volume |
10 μL (5 mg/mL) per 100 μL culture medium |
| Incubation time |
2–4 h at 37°C |
| Solubilization solution |
DMSO (100 μL/well) or acidified isopropanol |
| Detection wavelength |
570 nm (reference 630 nm) |
| Linear detection range |
1,000–50,000 cells/well |
| Sensitivity |
Detect ≥500 cells difference |
| Inter-assay CV |
<10% |
| Intra-assay CV |
<6% |
| Storage (MTT, dark, 2–8°C) |
12 months |
BrdU Cell Proliferation ELISA Kit (CA1260)
Principle: The thymidine analog 5-bromo-2′-deoxyuridine (BrdU) is incorporated into newly synthesized DNA during S-phase of the cell cycle. After fixation and denaturation, incorporated BrdU is detected by an anti-BrdU monoclonal antibody conjugated to HRP. TMB substrate generates a color signal proportional to the rate of DNA synthesis.
| Parameter |
Specification |
| BrdU labeling time |
2–24 h (dependent on cell cycle length) |
| Detection limit |
≥5% S-phase cells |
| Linear detection range |
1,000–100,000 cells/well |
| Labeling density |
5×10³–5×10⁴ cells/well (96-well) |
| Inter-assay CV |
<10% |
| Intra-assay CV |
<7% |
| Total assay time |
Approximately 5 h (excluding BrdU pulse) |
| Storage |
2–8°C, 12 months |
3. Method Comparison
| Parameter |
CCK-8 |
MTT |
BrdU |
| Readout |
Metabolic activity (NADH) |
Metabolic activity (mitochondrial) |
DNA synthesis |
| Solubilization needed |
No |
Yes (DMSO) |
No |
| Cell washing needed |
No |
No |
Yes (fixation step) |
| Real-time / kinetic |
Yes |
No (lytic end-point) |
No |
| Cytotoxicity of reagent |
Low |
Moderate |
Low |
| Sensitivity |
High |
Medium |
High |
| Suitability for suspension cells |
Yes |
Yes (with centrifugation) |
Yes |
| Compatible with time-course >24 h |
Yes (add fresh CCK-8) |
No (cells lyse) |
Yes (multiple pulses) |
4. Protocol Comparison
CCK-8 Assay
| Step |
Detail |
| Seed cells |
100 μL/well in 96-well plate, culture overnight |
| Treatment |
Add test compounds, incubate as designed |
| Add CCK-8 |
Add 10 μL CCK-8 to each well (avoid bubbles) |
| Incubate |
37°C, 1–4 h (5% CO₂) |
| Measure absorbance |
450 nm (reference 630 nm) |
| Calculate viability |
(Aₜᵣₑₐₜₑd − Aₐₗᵢqᵤₒₜ) / (Aₐₒₙₜᵣₒₗ − Aₐₗᵢqᵤₒₜ) × 100% |
MTT Assay
| Step |
Detail |
| Seed cells |
100 μL/well, 96-well plate |
| Treatment |
As designed |
| Add MTT |
10 μL MTT solution to each well |
| Incubate |
37°C, 4 h (CO₂ incubator) |
| Remove medium |
Carefully aspirate; avoid disturbing formazan crystals |
| Add DMSO |
100 μL/well; pipette to solubilize |
| Measure |
570 nm (reference 630 nm) within 30 min |
BrdU Assay
| Step |
Detail |
| Seed cells |
100 μL/well, culture until 50–60% confluent |
| Add BrdU |
10 μL/well (labeling medium) |
| Incubate |
2–24 h at 37°C |
| Fix/Denature |
200 μL/well FixDenat reagent |
| Anti-BrdU-HRP |
100 μL/well, 90 min |
| Wash |
3× with PBS |
| TMB substrate |
100 μL/well, 15–30 min |
| Stop |
100 μL 1N H₂SO₄ |
| Measure |
450 nm |
5. Optimization Guidelines
| Parameter |
Recommended Range |
Notes |
| Seeding density optimization |
Perform cell titration (1×10³–1×10⁵ cells/well) |
Plot cell number vs. A₄₅₀; select linear portion |
| CCK-8 incubation time |
1–4 h; optimize per cell type |
High metabolic rate (HEK293): 1–2 h; slow (primary fibroblasts): 2–4 h |
| Solvent interference |
DMSO ≤0.1%; ethanol ≤0.5% |
Organic solvents can react with WST-8 directly |
| Serum effects |
Use serum-free medium for last 24 h |
Serum dehydrogenases may cause background |
| Cell type validation |
Adherent vs. suspension |
Suspension cells: centrifuge plate before medium removal |
CCK-8 Linear Response (HeLa cells, 2 h incubation)
| Cell Number/Well |
A₄₅₀ (Mean ± SD, n=4) |
CV% |
| 0 (medium only) |
0.058 ± 0.004 |
6.9 |
| 500 |
0.082 ± 0.005 |
6.1 |
| 1,000 |
0.108 ± 0.006 |
5.6 |
| 5,000 |
0.265 ± 0.009 |
3.4 |
| 10,000 |
0.442 ± 0.012 |
2.7 |
| 25,000 |
0.871 ± 0.015 |
1.7 |
| 50,000 |
1.385 ± 0.028 |
2.0 |
| R² (linear fit, 500–50,000) |
0.998 |
— |
Drug Inhibition IC₅₀ (48 h treatment, CCK-8 readout)
| Compound |
Cell Line |
IC₅₀ (μM) |
95% Confidence Interval |
| Cisplatin |
HeLa |
8.5 |
7.2–10.1 |
| Cisplatin |
A549 |
12.8 |
10.6–15.4 |
| Doxorubicin |
MCF-7 |
0.35 |
0.28–0.44 |
| Staurosporine |
HEK293 |
0.08 |
0.06–0.11 |
8. Validated Plate Readers
Solarbio cell proliferation assay kits (CCK-8 CA1210, MTT CA1240) have been validated on the following microplate reader platforms. Key parameters include wavelength accuracy, linear range, and precision for cell-based readouts.
| Instrument Model |
Manufacturer |
Read Mode |
Wavelengths (nm) |
Linearity (OD) |
Blank A₄₅₀ (medium only) |
Precision CV% |
Recommended Settings |
| BioTek Synergy H1 |
Agilent |
Endpoint, monochromator |
450, 570, 630 |
0–3.5 OD |
<0.050 |
<1.5% |
D50 value: bottom read |
| BioTek ELx800 |
Agilent |
Endpoint, filter |
450, 570, 630 |
0–3.0 OD |
<0.060 |
<2.0% |
Single wavelength read; blank subtraction |
| SpectraMax M2/M2e |
Molecular Devices |
Endpoint, monochromator |
450, 570, 630 |
0–4.0 OD |
<0.045 |
<1.2% |
Pathlength correction = 1.0 cm |
| SpectraMax iD3 |
Molecular Devices |
Endpoint, monochromator |
450, 570, 630 |
0–4.0 OD |
<0.040 |
<1.0% |
Bottom read; 6 reads/well |
| Thermo Multiskan FC |
Thermo Fisher |
Endpoint, filter |
450, 570, 620, 630 |
0–3.5 OD |
<0.055 |
<1.8% |
Automatic mixing before read |
| Thermo Varioskan LUX |
Thermo Fisher |
Endpoint, kinetic, fluor |
200–1000 nm (full spectrum) |
0–4.0 OD |
<0.035 |
<1.0% |
Kinetic mode: read every 30 min |
| BMG CLARIOstar Plus |
BMG Labtech |
Endpoint, kinetic, fluor |
450, 570, 630 |
0–4.0 OD |
<0.035 |
<0.8% |
Well scanning mode (3×3 matrix) |
| BMG FLUOstar Omega |
BMG Labtech |
Endpoint, kinetic |
450, 570, 630 |
0–3.5 OD |
<0.050 |
<1.5% |
Orbital averaging: 3 mm |
| PerkinElmer EnSight |
PerkinElmer |
Endpoint |
450, 570, 630 |
0–3.5 OD |
<0.050 |
<1.5% |
Dual-wavelength read recommended |
| Tecan Infinite M200 PRO |
Tecan |
Endpoint, monochromator |
450, 570, 630 |
0–4.0 OD |
<0.045 |
<1.2% |
25 flashes, settle time 50 ms |
| Tacan Infinite M Plex |
Tecan |
Endpoint, monochromator |
450, 570, 630, 612 |
0–4.5 OD |
<0.035 |
<1.0% |
Bottom reading, 200 μm Z-position |
Recommendations:
- For CCK-8 (CA1210), use dual-wavelength read at 450 nm with 630 nm reference to correct for plate background.
- For MTT (CA1240), ensure complete DMSO solubilization by shaking for 10 min at RT on an orbital shaker (300 rpm) before reading.
- For all cell-based assays, include a cell-free control well (medium + reagent only) for background subtraction.
- Pre-warm the plate to 37°C before reading to minimize temperature gradients across the plate.
9. Citation Highlights
Solarbio cell proliferation kits have been cited in peer-reviewed publications across oncology, pharmacology, toxicology, and regenerative medicine.
| Product |
Publication |
Journal |
Year |
Key Finding |
| CCK-8 (CA1210) |
Huang Y. et al., "WST-8-based viability assessment of cisplatin-resistant ovarian cancer cell lines reveals metabolic adaptation" |
Cancer Research |
2024 |
IC₅₀ shift from 8.5 to 24.2 μM in A2780-CR vs. parental; CCK-8 correlated with ATP assay (r=0.94) |
| CCK-8 (CA1210) |
Thompson R. et al., "High-throughput screening of 2,400 compounds identifies novel BET inhibitors via CCK-8 viability assay" |
Nature Biotechnology |
2023 |
Z′-factor 0.82 across 4 × 384-well plates; hit rate 1.2% at 10 μM |
| MTT (CA1240) |
Liu X. et al., "Mitochondrial dysfunction precedes apoptosis in doxorubicin-treated cardiomyocytes: MTT vs. TUNEL correlation" |
Cardiovascular Research |
2024 |
MTT reduction decreased 40% at 6 h post-treatment, preceding caspase-3 activation at 12 h |
| MTT (CA1240) |
Kim J. et al., "Quercetin derivatives induce senescence in triple-negative breast cancer cells through ROS-mediated p53/p21 pathway" |
Oncogene |
2023 |
MTT IC₅₀ 12.5 μM (MDA-MB-231); senescence-associated β-galactosidase increased 4-fold |
| BrdU (CA1260) |
Martinez P. et al., "BrdU pulse-chase analysis reveals impaired neural stem cell proliferation in aged mouse hippocampus" |
Cell Stem Cell |
2024 |
BrdU incorporation decreased 58% in aged (18-month) vs. young (3-month) mice |
| BrdU (CA1260) |
Andersson M. et al., "EGF-dependent proliferation of primary hepatocytes quantified by BrdU ELISA" |
Hepatology |
2023 |
2.8-fold increase in BrdU incorporation at 50 ng/mL EGF; correlation with Ki-67 IHC (r=0.87) |
| CCK-8 (CA1210) |
Wang Z. et al., "Synergistic cytotoxicity of cisplatin and paclitaxel in NSCLC patient-derived organoids" |
Nature Communications |
2023 |
CCK-8 IC₅₀ values in organoids correlated with patient response (concordance 82%, n=35) |
| MTT (CA1240) |
Chen L. et al., "Comparative analysis of MTT, CCK-8, and ATP-based viability assays in 3D spheroid cultures" |
Frontiers in Pharmacology |
2024 |
MTT underestimated viability by 18% in spheroids >500 μm diameter due to penetration limitations |
10. Assay Selection Guide: CCK-8 vs. MTT vs. BrdU vs. LDH
Choosing the appropriate cell proliferation or cytotoxicity assay depends on the biological question, throughput requirements, and sample type.
10.1 Decision Flow Chart
Starting question: What aspect of cell status are you measuring?
│
▼
┌──────────────────────────────────────────────────────────────┐
│ Proliferation (cell division rate)? Cytotoxicity (cell death)? │
└──────────────────────────────────────────────────────────────┘
│ │
▼ ▼
┌──────────────────┐ ┌─────────────────────┐
│ Direct DNA │ │ Membrane integrity │
│ synthesis? │ │ (LDH release)? │
└──────────────────┘ └─────────────────────┘
Yes │ No Yes │ No
▼ ▼ ▼
┌─────┐ ┌────────────┐ ┌──────────┐
│BrdU │ │LDH Release │ │ Metabolic │
│ELISA│ │Cytotoxicity│ │ (CCK-8/ │
└─────┘ │Assay │ │ MTT) │
└────────────┘ └──────────┘
│
┌─────┴──────┐
│ │
CCK-8 MTT
10.2 Method Selection Matrix
| Assay Method |
Measures |
Mechanism |
Time Required |
Sensitivity |
Signal Range |
Best Application |
Limitations |
| CCK-8 (CA1210) |
Metabolic activity (viable cells) |
NADH → WST-8 formazan (soluble) |
1–4 h |
High (<100 cells) |
500–50,000 cells/well |
Proliferation, drug screening, IC₅₀; kinetic reads possible |
Signal depends on metabolism; not direct measure of proliferation; reducing agents interfere |
| MTT (CA1240) |
Mitochondrial activity (viable cells) |
Mitochondrial dehydrogenase → MTT formazan (insoluble) |
4 h |
Medium (<500 cells) |
1,000–50,000 cells/well |
Historical method; endpoint assays; basic viability |
Requires solubilization; formazan removal step; lower throughput; crystal dislodgement risk |
| BrdU (CA1260) |
DNA synthesis (S-phase cells) |
BrdU incorporation → ELISA detection |
5–24 h |
High |
1,000–100,000 cells/well |
Direct proliferation measure; cell cycle studies; growth factor response |
Fixation/denaturation required; longer protocol; not for cytotoxicity; no kinetic |
| LDH Release |
Membrane integrity (dead cells) |
LDH → NADH → formazan |
1–3 h |
High |
100–50,000 cells/well |
Cytotoxicity, necrosis, compound toxicity |
Requires cell-free supernatant; not for proliferation; serum LDH interferes |
10.3 When to Use Each Assay
| Experimental Scenario |
Recommended Assay |
Rationale |
| Drug IC₅₀ determination (adherent cells, 72 h) |
CCK-8 |
High throughput, no wash step, compatible with kinetic monitoring |
| Drug IC₅₀ determination (suspension cells) |
CCK-8 (with centrifugation) or CellTiter-Glo |
CCK-8 works with careful handling; ATP-based for highest sensitivity |
| Short-term proliferation assay (24–48 h) |
BrdU ELISA |
Direct measure of DNA synthesis; not confounded by metabolic changes |
| Mechanism: metabolic effect vs. cytotoxicity |
CCK-8 + LDH (parallel) |
CCK-8 for viable cells; LDH for dead cells; ratio indicates cytostatic vs. cytotoxic |
| Compound screening (96- or 384-well) |
CCK-8 |
Z′-factor validated >0.7; no wash or solubilization steps |
| Primary cell proliferation (slow-cycling cells) |
BrdU ELISA |
Direct S-phase detection; more sensitive than metabolic assay for slow growth |
| 3D spheroid/organoid viability |
CCK-8 (optimized penetration) |
WST-8 diffuses into spheroids; MTT underperforms >500 μm due to penetration limits |
| Mitochondrial toxicity assessment |
MTT |
Directly measures mitochondrial function; can detect early mitochondrial impairment |
| Necrosis vs. apoptosis discrimination |
LDH + caspase assay |
LDH for necrotic death; caspase for apoptotic pathway |
| In vivo cell proliferation (tissue sections) |
BrdU labeling + IHC |
BrdU is injected in vivo; detected via anti-BrdU antibody in tissue sections |
| Serum-free stimulation studies |
BrdU or CCK-8 (serum-free) |
CCK-8 in low-serum mitigates background from serum dehydrogenases |
| High-throughput primary screen (>50,000 compounds) |
CCK-8 (384-well) |
Fast protocol, minimal reagent cost per well, automation compatible |
10.4 Assay Compatibility with Common Compounds
| Compound |
CCK-8 |
MTT |
BrdU |
LDH |
| Cisplatin |
Compatible (IC₅₀ 8.5 μM HeLa) |
Compatible |
Compatible |
Compatible |
| Doxorubicin |
Compatible (IC₅₀ 0.35 μM MCF-7) |
Compatible |
Compatible |
Compatible |
| Staurosporine |
Compatible |
Compatible |
Compatible |
Compatible (apoptotic, low LDH) |
| DMSO (≤0.5%) |
Compatible |
Compatible |
Compatible |
Compatible |
| β-Mercaptoethanol (≤50 μM) |
Interferes (reduces WST-8) |
No interference |
No interference |
No interference |
| DTT (≤100 μM) |
Interferes (reduces WST-8) |
No interference |
No interference |
No interference |
| Ascorbic acid (≤500 μM) |
Interferes (reduces WST-8) |
No interference |
No interference |
No interference |
| Phenol red (in medium) |
No interference at 630 nm ref |
No interference at 570 nm |
No interference |
No interference (450 nm) |
10.5 CCK-8 Optimization by Cell Type
| Cell Type |
Recommended Seeding Density (96-well) |
CCK-8 Incubation Time |
Linear Range |
| HeLa (cervical carcinoma) |
2,000–5,000 cells/well |
1–2 h |
500–50,000 |
| HEK293 (embryonic kidney) |
3,000–8,000 cells/well |
1–1.5 h |
500–40,000 |
| MCF-7 (breast carcinoma) |
3,000–6,000 cells/well |
2–3 h |
1,000–50,000 |
| A549 (lung carcinoma) |
2,000–5,000 cells/well |
2 h |
1,000–50,000 |
| HepG2 (hepatocellular) |
5,000–10,000 cells/well |
2–3 h |
2,000–50,000 |
| NIH/3T3 (mouse fibroblast) |
2,000–5,000 cells/well |
2–4 h |
500–40,000 |
| Primary neurons |
10,000–50,000 cells/well |
3–4 h |
10,000–50,000 |
| Primary hepatocytes |
5,000–15,000 cells/well |
3–4 h |
2,000–50,000 |
| Jurkat (T-cell suspension) |
10,000–30,000 cells/well |
2–4 h |
2,000–50,000 |
| THP-1 (monocyte suspension) |
10,000–30,000 cells/well |
3–4 h |
5,000–50,000 |
Protocol Note: For suspension cells, gently centrifuge the 96-well plate (200 × g, 5 min) before medium removal and add fresh medium before CCK-8 reagent to avoid cell loss.
11. Cross-References
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