Annexin V-APC/PI Apoptosis Detection Kit, IC-6506
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 allophycocyanin (APC)-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. APC-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. 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-APC⁻/PI⁻), early apoptotic cells (Annexin V-APC⁺/PI⁻), late apoptotic cells (Annexin V-APC⁺/PI⁺), and necrotic cells (Annexin V-APC⁻/PI⁺). The APC fluorophore offers the advantage of excitation by standard red lasers (633-640 nm) with minimal spectral overlap with commonly used green fluorescent proteins and FITC, making this kit particularly suitable for multi-color flow cytometry panels. The kit includes all necessary reagents for a complete apoptosis detection workflow.
Product Features
1. APC-Labeled Annexin V: Bright, photostable allophycocyanin conjugate enables sensitive detection of PS externalization with excitation by standard red lasers.
2. Multi-Color Compatibility: APC fluorescence (Em ~660 nm) exhibits minimal spectral overlap with FITC, GFP, and PE channels, facilitating integration into multi-parameter flow cytometry panels.
3. Viability Discrimination: Propidium iodide co-staining enables simultaneous discrimination of viable, early apoptotic, late apoptotic, and necrotic cell populations.
4. Ready-to-Use Kit Format: Includes Annexin V-APC, Propidium Iodide, and 10¡Á Binding Buffer for a complete apoptosis detection workflow.
5. Broad Compatibility: Suitable for use with suspension and adherent cells from a wide range of species and cell types; compatible with standard flow cytometers equipped with a 633-640 nm laser.
Specifications
Size: Available in 50 assays and 100 assays
Detection Method: Flow cytometry
Fluorophores: Annexin V-APC (Ex/Em: 650/660 nm) and Propidium Iodide (Ex/Em: 535/617 nm)
Laser Lines: 633-640 nm (APC), 488 nm (PI)
Kit Components: Annexin V-APC, Propidium Iodide (PI), 10¡Á Binding Buffer
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 10¡Á Binding Buffer diluted to 1¡Á for staining. Do not use PBS or EDTA-containing buffers as they will chelate calcium and inhibit Annexin V binding. Protect APC-conjugated reagents from light.
1. Preparation of 1¡Á Binding Buffer: Dilute the provided 10¡Á Binding Buffer 1:10 with deionized water to obtain 1¡Á Binding Buffer (e.g., 1 mL of 10¡Á Binding Buffer + 9 mL of deionized water). Mix thoroughly. Prepare fresh before each use.
2. 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 1¡Á Binding Buffer at a concentration of 1¡Á10⁶ cells/mL.
3. Annexin V-APC Staining: Transfer 100 µL of the cell suspension (1¡Á10⁵ cells) to a flow cytometry tube. Add 5 µL of Annexin V-APC conjugate to each tube. Gently vortex and incubate at room temperature (25¡ãC) for 15 minutes in the dark.
4. Propidium Iodide Staining: After Annexin V-APC 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.
5. Dilution and Analysis: Add 400 µL of 1¡Á 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-APC only, and PI only). APC fluorescence is detected in the APC or Cy5 channel, and PI fluorescence is detected in the PE or PI channel.
Precautions
1. Annexin V binding requires calcium; always use the provided 10¡Á Binding Buffer diluted to 1¡Á for cell resuspension and staining. PBS and EDTA-containing buffers will inhibit binding.
2. Protect APC-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.
5. For research use only. Not for use in diagnostic or therapeutic procedures.
FAQ (Simplified)
Q1: What is the advantage of APC over FITC-labeled Annexin V?
A1: APC offers brighter fluorescence and superior photostability compared to FITC. Its excitation at 633-640 nm and emission at 660 nm minimizes spectral overlap with FITC, GFP, and PE, making it ideal for multi-color flow cytometry panels where green and yellow channels are occupied.
Q2: How should I set up compensation controls?
A2: Prepare single-stained control samples using cells induced to undergo apoptosis (e.g., by staurosporine treatment). Stain one sample with Annexin V-APC only and another with PI only. Include unstained cells as a negative control. Use these controls to set compensation between APC and PI channels.
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: Can I use this kit with GFP-expressing cells?
A4: Yes. The APC label on Annexin V emits in the far-red spectrum (~660 nm), which is well separated from GFP emission (~509 nm). This makes the kit particularly suitable for apoptosis detection in GFP-transfected or GFP-reporter cell lines without spectral overlap.
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-6506
Product Name: Annexin V-APC/PI Apoptosis Detection Kit
Size: 50 assays / 100 assays
Kit Components: Annexin V-APC, Propidium Iodide (PI), 10¡Á Binding Buffer
Price:
50 assays: CNY ¥1875.00 / USD $187.50 / EUR €225.00 / JPY ¥33750.00
100 assays: CNY ¥2900.00 / USD $290.00 / EUR €348.00 / JPY ¥52200.00
2023 Version
