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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 | APC |
Ex/Em | 650/660 |
Channel Set | APC |
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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1 Results
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1 Results
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Q1:Will using EDTA-containing trypsin affect Annexin V staining?
EDTA-containing trypsin can be used, but ensure thorough washing with PBS after digestion to remove residual EDTA.
Q2:Which Annexin V kit should be chosen for cell sample with green light?
If there is a DAPI channel in the flow cytometry, the E-CK-A258 Annexin V-APC/DAPI Apoptosis Detection Kit will be suitable. If not, the E-CK-A218 Annexin V-APC/7-AAD Apoptosis Detection Kit is avaliable.
Q3: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.
Q4:The treatment group samples were transfected with green fluorescence, so the Annexin V kit of APC/7-AAD was selected. How to set up the single positive control tubes?
Untransfected cells with obvious apoptosis were stained with 7-AAD and APC, respectively, as single positive control 1 and single positive control 2. The samples transfected with green fluorescence were directly put on the machine as single-positive control 3.
Q5: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.
Q6: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.
Q7: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.
Q8: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.Q9: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.
Q10: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.
