Annexin V-EGFP / PI Apoptosis Detection Kit

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      Catalog number:E-CK-A219

      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/PI 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

      Propidium Iodide (PI) is a common DNA dye that is not permeable to cell membrane. Once binding to DNA, the flurescence of PI increases by nearly 20 fold. Due to the loss of integrity of membrane, PI can enter late apoptotic or necrotic cells to stain DNA. Cells at different apoptotic stages can be distinguished by using Annexin V and PI.



      Components:



      Cat.

      Products

      20 T

      50 T

      100 T

      200 T

      Storage

      E-CK-A119

      Annexin V-EGFP

      100 μL

      250 μL

      500 μL

      1 mL

      2-8

      E-CK-A151

      Annexin V Binding Buffer (10 ×)

      1.4 mL×2

      5.5 mL

      11 mL

      11 mL×2

      2-8

      E-CK-A161

      Propidium Iodide (PI) Staining Solution

      100 μL

      250 μL

      500 μL

      1 mL

      2-8

      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 PI double-positive cells were necrotic or late apoptotic cells, and PI 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:

      one-step process

      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-EGFPand 5 µl of Propidium Iodide (PI) 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.


      Two-step process

      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 100 μL of 1 × Annexin V Binding Buffer to resuspend the cells.

      3. Add 2.5 µl of Annexin V-EGFPand 2.5 µl of Propidium Iodide (PI) Staining Solution to each tube. (Attributed to the higher resolution of two-step protocol, half the amount of the reagents can still guarantee a result of matched quality as in the one-step protocol. It's also recommended that users titrate the reagents for optimal performance in specific models.)

      4. Gently vortex the cells and incubate at room temperature for 15-20 min in the dark.

      5. Add 400 μL of 1 × Annexin V Binding Buffer to the tube, and mix gently.

      6. Analyze the cells immediately with proper machine settings. Otherwise, place the cells on ice in the dark

      and analyze within 1 h.





      Storage:

      Store at 4°C for up to one year. Annexin V-EGFPand PI Staining Solution should be stored in dark.


      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 PI as negative control. As for compensation controls, plesase use drug-treated cells stained with either Annexin V-EGFP or PI.

      3. Annexin V-EGFP can be detected in FITC channel while PerCP/Cy5.5 channel is preferred to PE channel for PI detection due to the large compensation needed between FITC and PE channels.

      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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