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Description
The Annexin V-FITC/PI Apoptosis Detection Kit is a ready-to-use flow cytometry assay for quantifying apoptosis in live, intact cells. By pairing FITC-conjugated Annexin V with propidium iodide (PI), it resolves viable, early-apoptotic, and late-apoptotic/necrotic populations in a single stain — a reliable workflow for apoptosis detection by flow cytometry or fluorescence microscopy.
How Annexin V/PI staining detects apoptosis
In healthy cells, phosphatidylserine (PS) faces the inner leaflet of the plasma membrane. Early in apoptosis, PS translocates to the cell surface before membrane integrity is lost. Annexin V is a Ca2+-dependent phospholipid-binding protein (35–36 kDa) with high affinity for PS, so FITC-labeled Annexin V binds externalized PS and identifies early-apoptotic cells.
Propidium iodide (PI) cannot cross the intact membrane of viable or early-apoptotic cells, but enters once membrane integrity is lost, staining the nuclei of late-apoptotic and necrotic cells. Used together, Annexin V and PI separate four populations in a single measurement: viable (Annexin V−/PI−), early apoptotic (Annexin V+/PI−), late apoptotic or necrotic (Annexin V+/PI+), and damaged or necrotic (Annexin V−/PI+).
Key features
- All reagents supplied in ready-to-use liquid form — no reconstitution required.
- Validated for both flow cytometry and fluorescence microscopy.
- Clear four-quadrant resolution of viable, early-apoptotic, and late-apoptotic/necrotic cells.
- Bright, well-separated FITC and PI signals for straightforward gating and imaging.
Result interpretation
| Staining pattern | Cell population |
| Annexin V− / PI− | Viable cells |
| Annexin V+ / PI− | Early apoptotic cells |
| Annexin V+ / PI+ | Late apoptotic / necrotic cells |
| Annexin V− / PI+ | Necrotic or mechanically damaged cells |
Annexin V/PI staining protocol (flow cytometry)
- Harvest cells gently (Accutase is preferred over trypsin) and avoid mechanical stress. Collect approximately 1–5 × 105 cells per sample.
- Wash cells twice with cold PBS, then resuspend in 1× Binding Buffer.
- Add Annexin V-FITC and PI Staining Solution according to the kit manual (typically 5 µL Annexin V-FITC and 5–10 µL PI per test); mix gently.
- Incubate for 10–15 min at room temperature, protected from light.
- Add 1× Binding Buffer and analyze by flow cytometry within 1 hour (488 nm excitation; FITC detected at ~530 nm, PI at ~617 nm). Do not fix cells before Annexin V staining.
Include unstained, Annexin V-only, and PI-only single-stain controls to set compensation and gating. Refer to the product manual for the full validated protocol.
Apoptosis assay comparison
| Method | What it detects | Best suited for |
| Annexin V / PI (this kit) | Phosphatidylserine externalization (early) and loss of membrane integrity (late) | Live, intact cells by flow cytometry or fluorescence microscopy |
| TUNEL | DNA fragmentation (late apoptosis) | Fixed cells and tissue sections |
| Caspase-3/7 activity | Effector caspase activation | Mechanistic confirmation of the apoptotic pathway |
Specifications
| Assay type | Annexin V-FITC / PI apoptosis detection (flow cytometry, fluorescence microscopy) |
| Detection targets | Viable, early-apoptotic, and late-apoptotic / necrotic cells |
| Fluorophores | Annexin V-FITC (Ex 488 nm / Em ~530 nm); propidium iodide (Em ~617 nm) |
| Sample type | Live, intact cultured cells (single-cell suspension) |
| Kit sizes | 20 T / 50 T / 100 T |
| Packing form | Bottle |
| Storage | −20 °C |
| Shipping | Dry ice |
| Shelf life | 12 months under recommended storage |
| Grade | For Research Use Only (RUO) |
Kit components
| Component No. | Name | 40302ES20 (20T) | 40302ES50 (50T) | 40302ES60 (100T) |
| 40302-A | Annexin V-FITC | 100 µL | 250 µL | 500 µL |
| 40302-B | PI Staining Solution | 200 µL | 500 µL | 1.0 mL |
| 40302-C | 1× Binding Buffer | 10 mL | 25 mL | 50 mL |
Storage & shipping
Shipped on dry ice. Store at −15 °C to −25 °C (−20 °C nominal); stable for 1 year under recommended conditions. Aliquot to minimize repeated freeze–thaw cycles.
What is the principle behind Annexin V-based apoptosis detection?
During early apoptosis, phosphatidylserine (PS) translocates from the inner to the outer leaflet of the plasma membrane before membrane integrity is lost. Annexin V binds PS with high affinity in a calcium-dependent manner, enabling early-apoptosis detection. Co-staining with propidium iodide (PI) discriminates: Annexin V⁺/PI⁻ = early apoptosis; Annexin V⁺/PI⁺ = late apoptosis/necrosis; Annexin V⁻/PI⁻ = viable cells.
What is the TUNEL assay and when should it be used instead of Annexin V?
TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) detects DNA strand breaks — a late-stage hallmark of apoptosis — by enzymatically labeling 3′-OH termini with fluorescent dUTP. TUNEL is preferred for detecting apoptosis in fixed cells, tissue sections, or when intracellular markers are needed (e.g., co-staining with nuclear markers). Annexin V is suitable for live, intact cells analyzed by flow cytometry or fluorescence microscopy.
Which fluorescent channels does this apoptosis kit require on a flow cytometer?
Channel requirements depend on the kit format: FITC variants require a 488 nm laser with 530/30 nm BP filter; YSFluor 488 variants are similar; YSFluor 647 requires a 633/647 nm (red) laser with 660/20 nm or 670 LP filter. Confirm your cytometer's laser and filter configuration matches the kit's excitation/emission profile. PI (propidium iodide) is typically detected in the PE or PerCP channel (488 nm excitation, 610–620 nm emission).
How should cells be prepared to avoid false-positive results?
Avoid mechanical or chemical stress during harvest — use gentle enzymatic dissociation (Accutase preferred over trypsin) and keep cells at 4°C during staining. Wash with cold Annexin V Binding Buffer (calcium-containing) immediately before staining. Do not fix cells before Annexin V staining — fixation disrupts membrane phospholipid asymmetry. Process and analyze samples within 1 hour of staining to prevent signal drift.
Still have a question about this kit?Our scientific team is happy to help with protocols, specs, or custom formats.
Contact UsYeasen Biotechnology offers flexible customization options for many of its assay kits and detection reagents, including custom lot sizes, bulk ordering, and application-specific formulation adjustments. Volume pricing, custom packaging, and kit bundling may be available depending on the product and intended workflow. A Certificate of Analysis (CoA) and lot-specific QC data are provided with every order. For inquiries regarding large-volume orders, custom configurations, or integration into automated workflows, please contact the BioHippo team for a tailored quotation.
