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G1514-50T / 100T Annexin V-IF647/PI Cell Apoptosis Detection Kit

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G1514-50T / 100T Annexin V-IF647/PI Cell Apoptosis Detection Kit

Apoptosis is a normal physiological process that occurs in the process of embryonic development and maintenance of tissue homeostasis. It is accompanied by many morphological changes. Among them, the loss of cell membrane is one of the early characteristics of apoptosis. In normal cells, phosphotidylserine (PS) is only distributed on the inner side of the phospholipid bilayer of the cell membrane, but in the early stage of apoptosis, PS will flip from the inner side of the lipid membrane to the outer side, exposing it to the outside of the cell. Annexin V (Annexin V) is a Ca2+-dependent phospholipid-binding protein with high affinity for PS and specifically binds to cells exposed to PS. Therefore, Annexin V is used as one of the indicators for detecting early cell apoptosis. Propidium Iodide (PI) is a nucleic acid dye. It cannot penetrate normal cells with intact cell membranes and early apoptotic cells, but can penetrate the cell membranes of late apoptotic and necrotic cells and stain the nucleus.

 

This product uses the IF647 fluorescent dye molecule labeled Annexin V as a detection probe to detect early cell apoptosis. At the same time, PI is used to distinguish surviving cells from necrotic and late apoptotic cells. Combined use of Annexin V-IF647 and PI, live cells show negative staining (Annexin V-/PI-), early apoptotic cells show single fluorescence positive (Annexin V+/PI-), and late apoptotic cells and necrotic cells show dual fluorescence Positive (Annexin V+/PI+). This kit is suitable for flow cytometry or fluorescence microscope detection.

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Product Variation:

Item code Description Volume
G1514-50T Annexin V-IF647/PI Cell Apoptosis Detection Kit 50T
G1514-100T 100T

 

Package Contents:

Item code Description G1514-50T G1514-100T
G1514-1 Annexin V-IF647 250µL 2x250µL
G1514-2 Propidium Iodide(PI) 250µL 2x250µL
G1514-3 1×Binding Buffer 25mL 2x25mL
  Uer Manual 1pc 1pc

Technical Specification

Storage and transportation

Transport in wet ice; store at 2-8°C in the dark, valid for 12 months..

 

 

Operation Steps:

  1. Suspension cells: Take the cell suspension, and collect the cells by centrifugation at 500 g for 5 min at 4°C;
    Adherent cells: first collect the cell culture supernatant. After digestion with EDTA-free trypsin (G4002 recommended), combine with the cell culture supernatant, and collect the cells by centrifugation at 500 g for 5 min at 4°C. Trypsin digestion time should not be too long, so as to avoid excessive digestion and cause false positives.
  2. Wash the cells twice with pre-cooled PBS (G4202 recommended), and collect the cells by centrifugation at 500 g each time for 5 min at 4°C;
  3. Gently resuspend the cells in the pre-chilled 1×Binding Buffer and adjust the cell concentration to 1~5×106/mL;
  4. Take 100 μL of cell suspension, add 5 μL Annexin V-IF647 and 5 μL PI, mix gently, and protect from light at room temperature for 8-10 min;
  5. Add 400 µL of pre-chilled 1×Binding Buffer, shake gently, and perform detection with a flow cytometer or fluorescence microscope within 1 h.
     

Result analysis:

  1. Flow Cytometry
    a. Select the appropriate voltage and adjust the light compensation during flow cytometry detection and analysis. It is recommended to set up a negative control (without any markers) for adjusting the voltage and single-label control (only Annexin V-IF647 and Annexin V-IF647 are added) except for the experimental group. Only add PI) for adjustment and compensation

    b. Reference examples for flow cytometry detection and analysis:
    Jurkat T lymphoma cells were induced with 5 µM Camptothecin for 6 h. Refer to the above experimental procedure and use flow cytometry to detect the results. The results are shown in the figure below.

    The maximum excitation wavelength of IF647 is 656 nm and the maximum emission wavelength is 670 nm; the maximum excitation wavelength of PI-DNA complex is 535 nm and the maximum emission wavelength is 615 nm. The two-dispersion point diagram is drawn by the relevant analysis software of the flow cytometer, and the IF647 is on the abscissa and the PI is on the ordinate. In a typical experiment, living cells have no fluorescence, and the scattered points are in the first quadrant of the lower left. The apoptotic cells in the early stage have strong pink fluorescence, and the scattered points are located in the second quadrant of the lower right. Late apoptotic cells and necrotic cells present pink and red double fluorescence, and the scattered points are located in the third quadrant of the upper right.
     
  2. Fluorescence microscope inspection
    Add 6 µL Annexin V-IF647 and PI double-stained cell suspension on the glass slide, cover it with a cover glass, and observe under a fluorescence microscope with a two-color filter. Annexin V-IF647 has a pink fluorescence signal and PI is red Fluorescence signal.

 

Notes:

  1. During the entire experiment, the operation should be as gentle as possible to avoid cell breakage and affect the experimental results.
  2. Washing the cells with PBS cannot be omitted, and at the same time, remove the residual PBS as much as possible.
  3. When using trypsin to digest cells, you should be careful to avoid artificial damage to the cells and control the digestion time. If the digestion time is too short, the cells need to be blown off to fall off, which is likely to cause mechanical damage to the cell membrane; if the digestion time is too long, the cell membrane is also vulnerable to damage. Affect the test results. In addition, pancreatin containing EDTA cannot be used. EDTA will affect the binding of Annexin V and PS.
  4. After the adherent cells are stimulated by apoptosis, if some of the cells float, it is necessary to collect the cell culture supernatant and the adherent cells and stain them at the same time, which will make the results more accurate.
  5. Annexin V-IF647 and PI are sensitive to light, please avoid light during operation. The test should be carried out as soon as possible after the reaction.
  6. Please wear laboratory clothes and disposable gloves during the experiment to avoid contamination and ensure safety.

 

This product is for scientific research purposes only, not for clinical diagnosis!

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