Annexin V-AF6477-AAD Cell Apoptosis Assay Kit
15-minute rapid staining; clear cell clustering and easy compensation adjustments.
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Instruction manual- Product Description
- Composition
- Precautions
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Item number and specifications
Item number Specifications 100-103-30 30 times 100-103-60 60 times 100-103-100 100 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 exhibits a narrower emission spectrum than PI and a longer emission wavelength, 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 Annexin V labeled with the fluorescent dye AF647 as a probe for detecting phosphatidylserine, in combination with 7‑AAD. Using a flow cytometer, fluorescence microscope, or other fluorescence‑based detection instruments, cell apoptosis can be rapidly assessed. Consequently, co‑staining with Annexin V‑AF647 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‑AF647 and 7‑AAD are normal; cells positive for Annexin V‑AF647 but negative for 7‑AAD represent early apoptotic cells; and cells positive for both Annexin V‑AF647 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‑AF647 and 7‑AAD solutions must be stored away from light.
Detection method
(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–AF647 or 7-AAD, used to adjust fluorescence channel compensation.
4. Pipette 100 μL of the cell suspension (with a total cell count of 1-5×10 5 cells) into a new EP tube, add 5 μL of Annexin V-AF647, 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.
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 duration. 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), and discard the supernatant.
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–AF647 or 7-AAD, used to adjust fluorescence channel compensation.
5. Pipette 100 μL of the cell suspension (with a total cell count of 1-5×10 5 cells) into a new EP tube, add 5 μL of Annexin V-AF647, 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
Flow cytometry analysis
AF647 has a maximum excitation wavelength of 651 nm and a maximum emission wavelength of 665 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.
Frequently Asked Questions and Solutions
1. False positive
After staining, the negative control (cells not subjected to apoptosis induction) exhibits an excessively high proportion of Annexin V‑AF647/7‑AAD double‑positive cells. This may be due to low cell viability; therefore, 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, we suggest switching to a different cell line or rescuing the cells anew; freshly revived cells should be passaged 1–2 times before proceeding with experiments.
2. Annexin V-AF647 staining failed or the positive rate was low.
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‑AF647 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.
After washing the cell pellet with PBS, any residual liquid should be removed as thoroughly as possible. Phosphate ions in the remaining PBS can precipitate as calcium phosphate.
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Product ingredients
Component Name 100-103-30 100-103-60 100-103-100 Annexin V-AF647 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 staining with Annexin V–AF647 and 7-AAD, 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 trypsin digestion time is too short, cells must be vigorously pipetted to detach, which can easily damage the cell membrane and lead to excessive uptake of 7-AAD. 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‑AF647 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, 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 1,500 rpm (200×g) to wash away the platelets.
5. Annexin V-AF647 and 7-AAD are photosensitive substances; please avoid light exposure during handling.
6. For your safety and health, please wear a lab coat and disposable gloves while performing this procedure.
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