Annexin V-FITC/7-AAD Apoptosis Assay Kit
15-minute rapid staining; clear cell clustering and easy compensation adjustment.
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Instruction manual- Product Description
- Composition
- Precautions
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Item number and specifications
Item number Specifications 100-105-30 30 times 100-105-60 60 times 100-105-100 100 times Product Overview
This kit is designed for the rapid detection of apoptosis. In the early stages of apoptosis, phosphatidylserine (PS) on the inner leaflet of the plasma membrane translocates to the cell surface; during the mid-to-late phases, larger molecules such as 7‑Aminoactinomycin D (7‑AAD), a DNA‑binding dye, can penetrate the cell and stain the nucleus with red fluorescence. 7‑AAD has a narrower emission spectrum than PI and emits at longer wavelengths, resulting in less interference with other detection channels, making it an ideal alternative to PI in multicolor fluorescence assays.
Annexin V is a Ca²⁺‑dependent phospholipid‑binding protein that specifically recognizes phosphatidylserine. This kit employs green fluorescent FITC‑labeled Annexin V as a probe for detecting phosphatidylserine, in combination with 7‑AAD. Using a flow cytometer, fluorescence microscope, or other fluorescence‑based detection instruments, apoptotic cells can be rapidly identified. Consequently, co‑staining with Annexin V‑FITC and 7‑AAD allows differentiation of cells at various stages of apoptosis. On a two‑color flow cytometry dot plot, cells negative for both Annexin V‑FITC and 7‑AAD are normal; cells positive for Annexin V‑FITC but negative for 7‑AAD represent early apoptotic cells; and cells positive for both Annexin V‑FITC and 7‑AAD correspond to late apoptotic or necrotic cells.
Storage conditions
This product is shipped with ice packs; store protected from light at 2–8°C, with a shelf life of 12 months. Annexin V-FITC and 7-AAD Solution must be stored away from light.
(1) Sample Staining – Suspended Cells
1. Centrifuge to collect the desired cells (1500 rpm, 5 min);
2. Add pre-cooled PBS to gently resuspend the cells, centrifuge to pellet the cells, and wash twice in total.
3. Dilute the 10× Binding Buffer with deionized water to prepare a 1× Binding Buffer, then resuspend the cells in the 1× Binding Buffer and adjust the cell concentration to 1–5 ×. 106 cells/mL;
▲ Flow cytometry requires the setup of three control samples to adjust voltage and compensate for fluorescence:
Blank tube: Cells resuspended solely in 1× Binding Buffer, used to assess the level of autofluorescence and adjust the instrument’s voltage.
Single-staining tubes: Stained separately with Annexin V-FITC or 7-AAD, used to adjust fluorescence channel compensation.
4. Pipette 100 μL of the cell suspension (with a total cell count of 1–5 × ) 105 Cells) were transferred to a new EP tube, 5 μL of Annexin V-FITC was added and gently mixed; then, 5 μL of 7-AAD was added and gently mixed, followed by incubation in the dark at room temperature for 10–15 minutes.
5. Add 400 μL of 1× Binding Buffer and gently mix. After staining, analyze the sample by flow cytometry within 1 hour whenever possible.
(1) Sample Staining – Adherent Cells
1. Aspirate the cell culture medium into a new centrifuge tube, then gently wash the adherent cells once with pre‑cooled PBS. Add trypsin solution sufficient to cover the cells, gently rock the dish to ensure thorough contact between the enzyme and the cells, and incubate at room temperature for an appropriate period. Gently pipette up and down to detach the adherent cells.
2. Add the cell culture supernatant collected in the previous step to the cells, gently mix, transfer to a centrifuge tube, centrifuge (1500 rpm, 5 min), discard the supernatant, and collect the cells.
3. Add pre-cooled PBS to gently resuspend the cells, centrifuge to pellet the cells, and wash twice in total.
4. Dilute the 10× Binding Buffer with deionized water to prepare a 1× Binding Buffer, then resuspend the cells in the 1× Binding Buffer and adjust the cell concentration to 1–5 ×. 106 cells/mL;
▲ Flow cytometry requires the setup of three control samples to adjust voltage and compensate for fluorescence:
Blank tube: Cells resuspended solely in 1× Binding Buffer, used to assess the level of autofluorescence and adjust the instrument’s voltage.
Single-staining tubes: Stained separately with Annexin V-FITC or 7-AAD, used to adjust fluorescence channel compensation.
5. Pipette 100 μL of the cell suspension (with a total cell count of 1–5× 105 cells) Transfer to a new EP tube, add 5 μL of Annexin V-FITC, and gently mix; then add 5 μL of 7-AAD, gently mix again, and incubate in the dark at room temperature for 10–15 minutes.
6. Add 400 μL of 1× Binding Buffer and gently mix. After staining, analyze the sample by flow cytometry within 1 hour whenever possible.
Sample Analysis
1. Flow cytometry analysis
FITC is analyzed by flow cytometry using the FL1 channel (Ex/Em = 490/525 nm); 7-AAD has a maximum excitation wavelength of 545 nm and a maximum emission wavelength of 650 nm, and its far-red fluorescence can be detected using the FL3 channel of a flow cytometer or a fluorescence microscope equipped with a 650-nm long-pass filter.
2. Fluorescence Microscopy Analysis
Place a drop of the stained cell suspension onto a microscope slide, cover the cells with a coverslip, and examine under a fluorescence microscope. Under the fluorescence microscope, use a dual‑color filter: the Annexin V–FITC fluorescence appears green, while the 7-AAD fluorescence appears red.
▲For adherent cells, you can directly culture them on coverslips and induce differentiation.
Frequently Asked Questions and Solutions
1. False positive
In cells that have not undergone apoptosis induction (negative control), the proportion of Annexin V‑FITC/7‑AAD double‑positive cells is excessively high. This may be due to low cell viability; we recommend using trypan blue staining to assess cell viability, with the proportion of trypan‑blue‑positive cells in the negative control should be less than 5%. If cell viability is low, consider switching to a different cell line or rescuing the cells by re‑thawing and culturing them. Newly thawed cells should be passaged 1–2 times before proceeding with experiments.
2. Annexin V-FITC staining fails or yields a low positive rate.
First, it is necessary to verify whether the apoptosis-inducing agent used in the experiment can indeed induce apoptosis. This can be confirmed by setting up a positive control group (cells treated with a known apoptotic inducer). If Annexin V–FITC staining fails, the most common procedural cause is improper detachment of adherent cells during digestion. Annexin V binds to PS only in the presence of Ca²⁺; since the binding buffer contains Ca²⁺, trypsinization with EDTA‑containing solutions can interfere with staining.
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Product ingredients
Component Name 100-105-30 100-105-60 100-105-100 Annexin V-FITC 150 µL 300 µL 500 µL 7-AAD Solution 150 µL 300 µL 500 µL 10×Binding Buffer 3 mL 6 mL 10 mL -
Precautions
1. Before Annexin V-FITC and 7-AAD staining, do not use fixatives or permeabilizing agents that compromise cell membrane integrity.
2. Throughout the entire procedure, perform all steps as gently as possible; do not blow or tap the cells with force to avoid mechanical damage.
3. For adherent cells, the following points should be observed during the digestion process:
1) When inducing apoptosis in adherent cells, if floating cells are present, both the floating and adherent cells should be collected and combined for staining.
2) If the trypsinization time is too short, cells must be vigorously pipetted to detach, which can easily damage the cell membrane and lead to excessive 7-AAD uptake. Conversely, if the digestion time is too long, the cell membrane may also be damaged, potentially interfering with the binding of PS and Annexin V‑FITC on the cell surface.
3) Use trypsin that does not contain EDTA whenever possible, as EDTA can interfere with the binding of Annexin V to PS. If you use trypsin containing EDTA to digest the cells, be sure to wash the cells twice with PBS prior to staining to remove the EDTA.
4. If the sample is derived from blood, be sure to remove platelets, as they contain PS, which can bind to Annexin V and interfere with the experimental results. You may use an EDTA‑containing buffer and centrifuge at 1500 rpm (200×g) to wash away the platelets.
5. Annexin V-FITC and 7-AAD are photosensitive compounds; please avoid light during handling.
6. For your safety and health, please wear a lab coat and disposable gloves while performing this procedure.
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