Elabscience® Annexin V-EV500 is developed to identify apoptotic and necrotic cells.Annexin V is a member of the annexin family, which binds to phosphatidylserine (PS) in a calcium-dependent manner. Annexin V-EV500, the EV500-conjugated format, binds specifically to the PS on the outer leaflet apoptotic cell membrane and can be detected with flow cytometry or fluorescence microscopy.
Cells at different apoptotic stages can be distinguished by using Annexin V and membrane impermeable DNA dyes like Propidium Iodide (PI), 7 7-Amino Actinomycin D (7-AAD).
|Cat.||Products||50 Tests||100 Tests||200 Tests||500 Tests||Storage|
|E-CK-A135||Annexin V-EV500 Reagent||250 μL||500 μL||1 mL||1.25 mLx2||2~8°C|
Jurkat cells were treated with 5 μM Camptothecin and detected with this reagent and PI.
Jurkat cells were cultured with (Left) or without (Right) 5 μM Camptothecin for 4 h. Annexin V-EV500 single-positive cells were early apoptotic cells, Annexin V-EV500 and PI double-positive cells were necrotic or late apoptotic cells, and PI single-positive cells were naked nuclei.
1.Induce apoptosis of suspension cells with reagents of interest. Collect cell cultures, centrifuge at 300 g for 5 min and discard the supernatant. Add PBS to wash the cells and resuspend the cells gently followed by the cell counting.
Tip: This product is only validated in suspension cells. Good cell viability is the key to the experiment. When the adherent cells are used for apoptotic detection, treatments like digestion may increase the ratio of necrotic or apoptotic cells and cause uncontrollable effects on the experimental results. Please be aware！
2.Split the cell suspension into tubes, 1~5 × 105 cells for each, centrifuge at 300 g for 5 min and discard the supernatant. Add PBS to wash the cells and discard the supernatant. Add 500 μL of 1 × Annexin V Binding Buffer [E-CK-A151] to resuspend the cells.
4. Gently vortex the cells and incubate at room temperature for 15~20 min in the dark.
5. Analyze the cells immediately with proper machine settings. Otherwise, place the cells on ice in the dark and analyze within 1 h.
1.For maximal assay performance, this reagent should be used within 12 months. Avoid freeze / thaw cycles.
2.Detect apoptosis as soon as possible after staining to avoid the increase number of apoptosis or necrosis.
3. Avoid extended exposure of the samples to direct light to protect the fluorophores from quenching.
4.For your safety and health, please wear the lab coat and disposable gloves before the experiments.
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