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| Detection Principle | Annexin V is a member of the annexin family, which binds to phosphatidylserine (PS) in a calcium-dependent manner. The Annexin V-labeled fluorescein bind specifically to the PS on the outer leaflet apoptotic cell membrane and can be detected with flow cytometry or fluorescence microscope. Nuclear dyes (PI, DAPI, 7-AAD) have a high DNA binding constant and is efficiently excluded by intact cells. It is useful for DNA analysis and dead cell discrimination during flow cytometric analysis. Due to late apoptosis or loss of membrane integrity in necrotic cells, nuclear dyes can enter cells to stain DNA, and when used in combination with Annexin V, cells in different apoptotic stages can be distinguished. |
| Detection Method | Fluorometric method, Flow cytometry |
| Sample Type | Cell samples |
| Assay Time | 40 min |
| Detection Instrument | Flow Cytometer |
| Dye Type | PE/Elab Fluor®594 |
| Ex/Em | 495;565/620 |
| Channel Set | ECD |
| Other Reagents Required | PBS(E-BC-R187), Deionized water |
| Storage | This product can be stored at 2-8°C for 12 months with shading light. |
| Expiration date | 12 months |
| Shipping | Ice bag |
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Q1:When detecting adherent cells, it is necessary to collect suspended cells generated after inducing apoptosis and detect them together with subsequent adherent cells collected.
Because during the culture process, apoptotic cells do not stick firmly to the flask, and a large number of them float in the culture medium. For the accuracy of the results, it is usually recommended that the suspended cells be collected and tested together with the cells digested by trypsin when detecting apoptosis.
Q2:None of my samples showed obvious apoptosis. How can I induce apoptosis as a positive control?
The cells in the control group can be induced apoptosis by the following two methods: ① Since different cells are affected by DMSO to different degrees, it is recommended to explore the action time or volume fraction of DMSO through pre-experiment. The action time can be explored according to the following volume fraction (10%) first, and the microscopic examination is conducted every 30 minutes. ② The cells were bathed in a water bath at 50~60℃ for 5~10min. When the cell morphology changes or a large number of floating, can be considered to produce apoptotic cell population.
Q3:Is it able to detect blood samples with Annexin V assay kit?
It is not recommended to do so. Platelet and some other immune cells in blood may have exposed PS in normal state that will affect the test results.
Q4:Is Annexin V Kit able to detect cryopreserved cells directly?
It is not recommended to do so. Cryopreservation may lead to apoptosis and necrosis, resulting in inaccurate experimental results.
Q5:How to set the gate for rathippocampal neuron apoptosis assay?
Annexin V assay is normally used for cell line samples, thus only debris need to be excluded by gating. Since there aremultiple types of cells in tissue samples, it depends on the purpose of experiment, whether it means to detect apoptosis of the whole tissue sample or some specific cells. Cells can be gated by FSC/SSC or some other biomarkers.
Besides, operation of tissue sample preparation may also effect the result, Inappropriate operation may also result in false positives due to sample handling.Q6:Can the temperature of Annexin V be 4℃?
It is recommended to incubate at room temperature, about 25℃. The AnnexinV staining process is actually the process of binding the AnnexinV protein specifically to phosphatidylserine on the cell membrane, and this process is appropriate when the cell function and enzyme activity are normal.
Q7:Can the Binding buffer be replaced with PBS containing calcium ions?
No, the concentration of calcium ions in binding buffer is determined after experimental exploration, and PBS containing calcium ions may not have the effect of binding buffer.
Q8:Can PBS be used to replace Binding Buffer in Annexin V assay?
No, because Annexin V binds to phosphatidylserine (PS) dependent on Ca2+, which is not present in common PBS solutions, while Binding buffers contain Ca2+ to facilitate Annexin V binding to PS. Without Binding Buffer, Annexin V binding capacity will be reduced, resulting in significantly reduced positive signal or even no positive signal.
Q9:Can Annexin V kit be used on adherent cells?
Annexin V kit can be used on adherent cells, with the following caveat:
1. In late stage of apoptosis, cells may not able wo maintain adhesion, so floating cells should be collected for the test.
2. The dissociation of adherent cells should be handled gently to avoid cell damage caused by human operation.
3. Do not digest for too long with trypsin, avoid the usage of the trypsin contain EDTA. If EDTA-containing trypsin must be used, wash the cells to remove EDTA as much as possible.Q10:Can Annexin V induce apoptosis in mouse hippocampal tissue?
It is not advised to use tissue samples for Annexin V experiments. On the one hand, in the process of preparing tissue samples into single-cell suspension, the apoptosis rate caused by the experimental operation may be higher than that of the sample itself. Moreover, the experimental operation is unstable, and the actual apoptosis rate of the sample itself cannot be determined by the final experiment. On the other hand, tissue samples have multiple cell types, each with different autofluorescence, and the results cannot be analyzed.
