Technical Specification: Apoptosis Detection Kits¶

1. Product Range¶

Product	SKU	Method	Detection Platform	Assays per Kit
Annexin V-FITC Apoptosis Detection Kit	CA1020	Annexin V-FITC/PI dual stain	Flow cytometry, fluorescence microscopy	50–100 assays
TUNEL Apoptosis Assay Kit	CA1040	TdT-mediated dUTP nick end-labeling	Fluorescence microscopy	50 assays
Caspase-3 Activity Assay Kit	BC3830	DEVD-pNA cleavage, 405 nm	Microplate reader	100 assays
Annexin V-APC/PI Apoptosis Detection Kit	CA1025	Annexin V-APC/PI dual stain	Flow cytometry (APC channel compatible)	50–100 assays
Hoechst 33342/PI Double Stain Kit	CA1120	Hoechst 33342 + PI dual stain	Fluorescence microscopy	100 assays

2. Technical Parameters¶

Annexin V-FITC/PI Kit (CA1020)¶

Principle: Annexin V is a 35.8 kDa Ca²⁺-dependent phospholipid-binding protein with high affinity for phosphatidylserine (PS). In viable cells, PS is sequestered to the inner leaflet of the plasma membrane by flippase (an ATP-dependent aminophospholipid translocase). During early apoptosis, flippase is inactivated and scramblase is activated, causing PS externalization to the outer membrane leaflet. Annexin V-FITC binds exposed PS. Propidium Iodide (PI) is a membrane-impermeant nucleic acid dye that enters only cells with compromised plasma membranes (late apoptosis or necrosis).

The binding reaction:

PS(externalized) + Annexin V-FITC  →  PS-Annexin V-FITC complex
  (Ca²⁺-dependent, Kd ≈ 5 nM)

Parameter	Specification
Cell preparation	1×10⁵–5×10⁵ cells per assay
Staining time	15 min at room temperature (dark)
Detection wavelengths	FITC Ex/Em: 488/525 nm; PI Ex/Em: 535/615 nm
Binding buffer	10 mM HEPES/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂
Viable cells	Annexin V⁻, PI⁻
Early apoptosis	Annexin V⁺, PI⁻
Late apoptosis/necrosis	Annexin V⁺, PI⁺
Necrosis (primary)	Annexin V⁻, PI⁺
Kit storage	2–8°C, 12 months
Detection limit	≥1% apoptotic cells in population

TUNEL Assay Kit (CA1040)¶

Principle: During apoptosis, endonucleases (primarily CAD — caspase-activated DNase) cleave genomic DNA at internucleosomal linker regions, generating double-strand breaks with 3′-hydroxyl (3′-OH) ends. Terminal deoxynucleotidyl transferase (TdT) catalyzes the template-independent incorporation of fluorescein-dUTP onto these 3′-OH ends. The incorporated fluorescein can then be detected by fluorescence microscopy or flow cytometry.

The enzymatic reaction:

DNA(3′-OH ends) + Fluorescein-dUTP  →  DNA-3′-Fluorescein-dUMP + PPi
              ↑                          ↑
            TdT enzyme        Catalyzed by TdT

Parameter	Specification
Sample type	Fixed cells, paraffin-embedded tissue sections
Fixation	4% paraformaldehyde, 15 min at RT
Permeabilization	0.1% Triton X-100 in 0.1% sodium citrate, 2 min on ice
TdT incubation	60 min at 37°C (dark, humidified)
Detection	Fluorescein Ex/Em: 488/525 nm
Counterstain	DAPI or PI
Positive control	DNase I-treated sample (10 U/mL, 10 min at RT)
Kit storage	-20°C, 12 months

Caspase-3 Activity Assay Kit (BC3830)¶

Principle: Caspase-3 (CPP32/apopain), a 32 kDa proenzyme classified as an executioner caspase, is proteolytically cleaved at Asp175-Ser176 and Asp28-Ser29 to generate active p17 and p12 subunits during apoptosis. The kit uses a synthetic tetrapeptide substrate, DEVD-pNA (Asp-Glu-Val-Asp conjugated to p-nitroaniline). Active caspase-3 cleaves the DEVD-pNA substrate, releasing free pNA, which absorbs at 405 nm.

The enzymatic reaction:

Ac-DEVD-pNA  →  Ac-DEVD-OH + pNA
        ↑                              ↑
    Caspase-3             Free pNA (absorbs at 405 nm)

Parameter	Specification
Substrate	Ac-DEVD-pNA (200 μM final concentration)
Detection wavelength	405 nm (reference 630 nm)
Incubation time	1–2 h at 37°C
Protein input	20–100 μg total protein per assay
LOD (recombinant caspase-3)	~1 U/mL
Linear detection range	1–50 U/mL recombinant caspase-3
Intra-assay CV	<8%
Kit storage	-20°C, 12 months
Total assay time	Approximately 2.5 h

3. Performance Data — Representative Caspase-3 Activity Standard Curve¶

Recombinant Caspase-3 (U/mL)	A₄₀₅ (Mean ± SD, n=3)	CV%
0 (blank)	0.025 ± 0.002	8.0
0.5	0.032 ± 0.002	6.3
1	0.042 ± 0.003	7.1
2.5	0.076 ± 0.004	5.3
5	0.135 ± 0.005	3.7
10	0.248 ± 0.008	3.2
25	0.512 ± 0.015	2.9
50	0.898 ± 0.022	2.5

4. Protocol Optimization Guidelines¶

Cell Treatment Duration for Apoptosis Induction¶

Inducer	Typical Concentration	Recommended Duration
Staurosporine (broad kinase inhibitor)	1 μM	4–6 h (early apoptosis)
Etoposide (topoisomerase II inhibitor)	10–50 μM	12–24 h
Doxorubicin (DNA intercalator)	1–10 μM	12–24 h
Actinomycin D (transcription inhibitor)	0.1–1 μg/mL	6–12 h
UV irradiation (254 nm)	50–100 J/m²	4–8 h post-exposure
Fas ligand (300 ng/mL) + cycloheximide	1 μg/mL	4–6 h
H₂O₂ (oxidative stress)	100–500 μM	2–6 h

Critical Considerations for Each Method¶

Method	Key Optimization Parameters	Common Pitfalls
Annexin V-FITC/PI	Ca²⁺ concentration in binding buffer; gentle resuspension	Excessive washing loses early apoptotic cells; prolonged incubation (>1 h) allows late-stage PI uptake
TUNEL	Permeabilization time; TdT concentration	Over-permeabilization causes false positives; insufficient TdT reduces sensitivity
Caspase-3 Activity	Protein input normalization; substrate saturating concentration	Degraded lysates lose activity; hemoglobin/heme interference in 405 nm read

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