Annexin V-EGFP / 7-AAD Apoptosis Detection Kit

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      Catalog number:E-CK-A220

      Size:
      • 20T
      • 50T
      • 100T
      • 200T
      Qty:
      - +
      Price: $75

      Lead Time: 7~10 daysWelcome to order from local distributors.

      Add to cart Compare Bulk request Manual


      Introduction:

      Elabscience® Annexin V-EGFP/7-AAD Cell Apoptosis Detection kit 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.  The fluorescent format of this intracellular protein, Annexin V-EGFP, binds specifically to the PS on outer leaflet of cell membrane by flow cytometry or fluorescence microscopy.

      7-AAD (7-amino-actinomycin D) has 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 the loss of integrity of membrane, 7-AAD can enter late apoptotic or necrotic cells to stain DNA. Cells at different apoptotic stages can be distinguished by using Annexin V and 7-AAD.



      Components:



      Cat.

      Products

      50 T

      100 T

      200 T

      Storage

       E-CK-A119

       Annexin V-EGFP

      250 μL

      500 μL

      1 mL

      2-8

       E-CK-A151

       Annexin V Binding Buffer (10 ×)

      10 mL

      10 mL

      10 mL

      2-8

       E-CK-A162

       7-AAD Viability Staining Solution

      250 μL

      500 μL

      1 mL

      -20

                                            Manual

       One Copy





      Jurkat cells were treated with 1μM Camptothecin and detected by this kit:





           Jurkat cells were treated with 1μM Camptothecin (Left) or not (Right) for 4 h. Annexin V-EGFP single-positive cells were early apoptotic cells, Annexin V-EGFP and 7-AAD double-positive cells were necrotic or late apoptotic cells, and 7-AAD single-positive cells were nude nuclear cells.



      Instructions:

      The Annexin V Binding Buffer (10 ×)[Cat:E-CK-A151] is a 10 × concentrated solution. Dilute with DI water to 1 × working solution before use.

      For example: Take 1 mL Annexin V Binding Buffer (10 ×), dilute with DI water to 10 mL.


      Staining Procedure:

      1. Induce apoptosis of suspension cells with reagents of interest. Collect cell cultures and centrifuge at 300 g for 5 min, discard the supernatant. Add appropriate PBS to wash the cells, resuspend gently and count the cells.

      Note: 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, discard the supernatant. Add appropriate PBS to wash the cells and discard the supernatant. Add 500 μL of 1 × Annexin V Binding Buffer to resuspend the cells.

      3. Add 5 µl of Annexin V-EGFP and 5 µl of 7-AAD Viability Staining Solution to each tube.

      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.


      Storage:

      Annexin V-EGFP and 7-AAD Viability Staining Solution should be stored in dark.

      7-AAD Viability Staining Solution should be split into small tubes and storage at -20°C.

      Other reagents should be stored at 4 °C.


      Cautions:

      1.  For maximal assay performance, this kit should be used within 12 months. Avoid freeze / thaw cycles.

      2. For FCM analysis, please set untreated cells stained with both Annexin V-EGFP and 7-AAD as negative control. As for compensation controls, plesase use drug-treated cells stained with either Annexin V-EGFP or 7-AAD.

      3. Annexin V-EGFP can be detected in FITC channel while 7-AAD can be detected in PerCP/Cy5.5 channel.

      4.  Detect apoptosis as soon as possible after staining to avoid the increase number of apoptosis or necrosis.

      5.  Avoid extended exposure of the samples to direct light to protect the fluorophores from quenching.

      6.  For your safety and health, please wear the lab coat and disposable gloves before the experiments.




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