Annexin V-FITC Apoptosis Detection Kit, IC-6501
Product Introduction
This product is a ready-to-use flow cytometry kit for the quantitative detection of apoptotic cells based on the high-affinity binding of fluorochrome-conjugated Annexin V to phosphatidylserine (PS) residues exposed on the outer leaflet of the plasma membrane. In healthy viable cells, PS is actively maintained on the inner leaflet of the plasma membrane by aminophospholipid translocase. During early apoptosis, this asymmetry is lost and PS becomes exposed on the cell surface, where it serves as an "eat-me" signal for phagocytic clearance. Fluoroscein isothiocyanate (FITC)-labeled Annexin V specifically binds to exposed PS in a calcium-dependent manner, enabling the identification of early and late apoptotic cells by flow cytometry or fluorescence microscopy. When used in combination with propidium iodide (PI), a membrane-impermeable nucleic acid dye that stains only late apoptotic and necrotic cells with compromised membrane integrity, this kit allows the discrimination of viable cells (Annexin V-/PI-), early apoptotic cells (Annexin V+/PI-), late apoptotic cells (Annexin V+/PI+), and necrotic cells (Annexin V-/PI+). The kit includes all necessary reagents, including binding buffer optimized for Annexin V staining, making it a convenient and reliable tool for apoptosis research.
Product Features
1. High Sensitivity Detection: FITC-labeled Annexin V provides bright green fluorescence for sensitive and specific detection of PS externalization in early apoptotic cells.
2. Viability Discrimination: Propidium iodide co-staining enables simultaneous discrimination of viable, early apoptotic, late apoptotic, and necrotic cell populations.
3. Ready-to-Use Kit Format: Includes all necessary reagents (Annexin V-FITC, Propidium Iodide, and Binding Buffer) for a complete apoptosis detection workflow.
4. Broad Compatibility: Suitable for use with suspension and adherent cells from a wide range of species and cell types; compatible with standard flow cytometry instruments equipped with a 488 nm laser.
5. Rapid and Simple Protocol: Single-step staining procedure requiring only 15-20 minutes of incubation, with no wash steps required before analysis.
Specifications
Size: Available in 50 assays and 100 assays
Detection Method: Flow cytometry or fluorescence microscopy
Fluorophore: Annexin V-FITC (Ex/Em: 494/518 nm) and Propidium Iodide (Ex/Em: 535/617 nm)
Laser Line: 488 nm (blue laser)
Sample Types: Suspension and adherent cells
Storage and Stability
Storage Conditions: Store all kit components at 4¡ãC, protected from light. Do not freeze.
Shelf Life: The product is stable for 12 months from the date of manufacture when stored as directed. After opening, use within 3 months when handled aseptically and stored at 4¡ãC.
Protocol (For Reference Only)
Important: Annexin V binding is calcium-dependent; always use the provided Binding Buffer for staining. Do not use PBS or EDTA-containing buffers as they will chelate calcium and inhibit Annexin V binding. Protect FITC-conjugated reagents from light.
1. Cell Preparation: Harvest cells by gentle trypsinization (adherent cells) or by centrifugation (suspension cells). Wash cells twice with cold PBS (without Ca²⁺/Mg²⁺) and centrifuge at 300 x g for 5 minutes. Resuspend cells in 1X Binding Buffer at a concentration of 1¡Á10⁶ cells/mL.
2. Annexin V-FITC Staining: Transfer 100 µL of the cell suspension (1¡Á10⁵ cells) to a 1.5 mL microcentrifuge tube or appropriate flow cytometry tube. Add 5 µL of Annexin V-FITC conjugate to each tube. Gently vortex and incubate at room temperature (25¡ãC) for 15 minutes in the dark.
3. Propidium Iodide Staining: After Annexin V-FITC incubation, add 5 µL of Propidium Iodide Solution to each tube. Gently vortex and incubate at room temperature for an additional 5 minutes in the dark. Do not wash cells.
4. Dilution and Analysis: Add 400 µL of 1X Binding Buffer to each tube, mix gently, and analyze immediately by flow cytometry. Acquire at least 10,000 events per sample. Set up proper compensation using single-stained control samples (unstained cells, Annexin V-FITC only, and PI only).
5. Data Analysis: Analyze flow cytometry data by creating a bivariate scatter plot of FITC (FL1-H) versus PI (FL2-H or FL3-H). Gate on the main cell population and quadrant the plot to distinguish viable cells (Annexin V-/PI-), early apoptotic cells (Annexin V+/PI-), late apoptotic cells (Annexin V+/PI+), and necrotic cells (Annexin V-/PI+).
Precautions
1. Annexin V binding requires calcium; always use the provided calcium-containing Binding Buffer for resuspension and staining. PBS and EDTA-containing buffers will inhibit binding.
2. Protect FITC-conjugated reagents from light throughout the staining procedure to prevent photobleaching.
3. Analyze samples immediately after staining; prolonged storage may lead to increased PI uptake and false-positive necrotic cell counts.
4. For adherent cells, use gentle trypsinization protocols to avoid membrane damage and false-positive Annexin V staining. Include appropriate controls to establish baseline fluorescence.
5. For research use only. Not for use in diagnostic or therapeutic procedures.
FAQ (Simplified)
Q1: Can I use PBS instead of the provided Binding Buffer?
A1: No. Annexin V binding to phosphatidylserine is strictly calcium-dependent. The provided Binding Buffer contains the optimal calcium concentration for high-affinity binding. PBS and EDTA-containing buffers will chelate calcium and significantly reduce or abolish Annexin V staining.
Q2: How should I set up compensation controls?
A2: Prepare single-stained control samples using cells that express phosphatidylserine (e.g., apoptotic cells induced by staurosporine or UV treatment). Stain one sample with Annexin V-FITC only and another with PI only. Use untreated viable cells as an unstained control.
Q3: Can I fix cells after staining?
A3: Fixation is not recommended as it may cause cell membrane permeabilization and affect PI exclusion, leading to altered staining patterns. If fixation is absolutely required, use 1% paraformaldehyde for no more than 10 minutes and analyze immediately.
Q4: What if my cell population shows high background Annexin V staining?
A4: Elevated background may result from membrane damage during cell harvesting, residual trypsin activity, or prolonged sample handling. Use gentle centrifugation speeds, ensure complete trypsin neutralization with serum-containing medium, and process samples promptly on ice.
Disclaimer
1. For Research Use Only. Not for use in diagnostic or therapeutic procedures.
2. Due to the variable nature of biological research, optimization of cell preparation and staining conditions is recommended for specific cell types and experimental treatments.
3. This warranty is limited to the replacement of the product. The manufacturer assumes no liability for incidental or consequential damages, including loss of samples or data.
4. Wear appropriate protective clothing and gloves when handling this product. Propidium Iodide is a potential mutagen; handle with appropriate safety precautions.
Ordering Information
Catalog Number: IC-6501
Product Name: Annexin V-FITC Apoptosis Detection Kit
Size: 20 assays / 50 assays / 100 assays
Price:
20 assays: CNY ¥950.00 / USD $95.00 / EUR €114.00 / JPY ¥17100.00
50 assays: CNY ¥1900.00 / USD $190.00 / EUR €228.00 / JPY ¥34200.00
100 assays: CNY ¥3150.00 / USD $315.00 / EUR €378.00 / JPY ¥56700.00
