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Description
Annexin V-EGFP/PI cell apoptosis detection kit uses EGFP-labeled Annexin V as a probe to detect the occurrence of early cell apoptosis.
The detection principle is as follows: in normal living cells, phosphotidylserine (PS) is located on the inner side of the cell membrane, but in early apoptotic cells, PS flips from the inner side of the cell membrane to the surface of the cell membrane and is exposed to the extracellular environment. Annexin-V is a Ca2+ -dependent phospholipid-binding protein with a molecular weight of 35 to 36 kDa that binds to PS with high affinity. The phosphatidyl serine can bind to the membrane of early apoptotic cells through the exposed phosphatidyl serine on the outside of the cell.
In addition, Propidium Iodide (PI) is provided to distinguish surviving early cells from necrotic or late apoptotic cells. PI is a nucleic acid dye, which can not pass through the intact cell membrane of normal cells or early apoptotic cells but can pass through the cell membrane of late apoptotic and necrotic cells and make the nucleus red. Thus, when Annexin V was combined with PI, PI was excluded from living cells (Annexin V-/PI-) and early apoptotic cells (Annexin V+/PI-). The late apoptotic and necrotic cells were double positive for both EGFP and PI (Annexin V+/PI+).
This kit is suitable for flow cytometry and fluorescence microscopy.
Product Components
|
Component |
|
40303ES20（20 T） |
40303ES50（50 T） |
40303ES60（100 T） |
|
40303-A |
Annexin V-EGFP |
0.1 mL |
0.25 mL |
0.5 mL |
|
40303-B |
PI Staining Solution |
0.2 mL |
0.5 mL |
1 mL |
|
40303-C |
1×Binding Buffer |
10 mL |
25 mL |
50 mL |
Shipping and Storage
The components are shipped with an ice pack and can be stored at -20°C keep out of light, and avoid repeated freezing and thawing for 1 year.
[Notes]: If it needs to be used repeatedly in a short period, it can be stored at 4 ℃ and protected from light for half a year.
Cautions
1) Since apoptosis is a rapid process, it is recommended that samples be analyzed within 1 hour after staining.
2) For adherent cells, digestion is a critical step. Do not use EDTA in the digestive solution as EDTA can affect the binding of Annexin V to PS.
3) Annexin V-FITC, but not Annexin V-EGFP, should be used to fix cells, such as to detect apoptosis and cell cycle at the same time, because EGFP would be denatured and lose its ability to excite fluorescence during fixation. Cells were incubated with Annexin V-FITC before fixation and unbound Annexin V-FITC was washed away with Binding Buffer. Because increased cell permeability during fixation creates cell debris that can bind to Annexin V and interfere with the results.
4) If the sample is from blood, be sure to remove platelets from the blood. Because platelets contain PS, which can bind to Annexin V, interfering with the results. Platelets can be washed away by using a buffering agent containing EDTA and centrifuging at 200 g.
5) Please centrifuge the reagent briefly before opening the cover, and throw the liquid on the inner wall of the cover to the bottom of the tube to avoid liquid sprinkling when opening the cover.
6) Annexin V-EGFP and PI are photosensitive substances, so please avoid light during operation.
7) For research use only!
Instructions
Experiment design
Blank tube: Negative control cells without Annexin V-EGFP and PI Staining Solution were used for voltage regulation.
Single stained tubes: Positive control cells, supplemented only with Annexin V-EGFP, were used to modulate compensation.
Detecting tube: The cells were treated with Annexin V-EGFP and PI Staining Solution. The experimental data were obtained by adjusting the parameters of the blank tube and single dye tube.
1.1 Sample dyeing
1) suspension cell: Cells were collected by centrifugation at 300 g for 5 min at 4 ° C.
adherent cell: After digestion with trypsin without EDTA, cells were harvested by centrifugation at 300 g for 5 min at 4 ° C. Trypsin digestion time should not be too long to prevent false positives.
2) The cells were washed with precooled PBS twice, each time at 300 g, and centrifuged at 4 ℃ for 5 min.
3) The cells were resuspended with 1×Binding Buffer and the concentration was adjusted to 1~5 ×106/mL.
4) 100 μL of cell suspension was added into 5 mL flow cytometry tubes, 5 μl Annexin V-EGFP was added, and the cells were mixed and incubated for 5 min at room temperature under the light.
5) Adding 10 μl PI Solution and 400 μl PBS, the Staining was performed immediately.
[Notes]: When using flow cytometry to detect apoptosis, PI is greatly affected by time, and too long time will lead to an increase in PI staining, so flow cytometry should be completed within 1 h.
1.2 Flow cytometry analysis
The maximum excitation wavelength of EGFP was 488 nm, and the maximum emission wavelength was 507 nm; The maximum excitation wavelength of the PI-DNA complex was 535 nm, and the maximum emission wavelength was 615 nm. Software such as CellQuest was used for analysis and a two-color dot plot was drawn, EGFP is the abscissa and PI is the ordinate. Collect 10,000 events per sample. In a typical experiment, the cells can be divided into three subgroups, living cells only have very low-intensity background fluorescence, early apoptotic cells only have strong green fluorescence, and late apoptotic cells have green and red fluorescence double staining.
Documents:
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.
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.
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).
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.
Yeasen 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.
