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Annexin V-IF488/PI Cell Apoptosis Detection Kit

Volume:50T
  • G1513-50T
  • Servicebio
  • Servicebio

Product Description

Product information:

 

Product name

Cat. No.

Volume

Annexin V-IF488PI Cell Apoptosis Detection Kit

G1513-50T

50T

G1513-100T

100T

 

Product description:

 

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 IF488 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. When Annexin V-IF488 is used in combination with PI, living 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.

 

Storage and transportation

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

 

Component:

 

Component Number

Component

G1513-50T

G1513-100T

G1513-1

Annexin V-IF488

250 μL

2×250 μL

G1513-2

Propidium Iodide(PI)

250 μL

2×250 μL

G1513-3

1×Binding Buffer

25 mL

2×25 mL

Product User Manual

1pc

 

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-IF488 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 a negative control (without any markers) to adjust the voltage, except for the experimental group, and a single-label control (only Annexin V-IF488 and 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 IF488 is 491 nm and the maximum emission wavelength is 518 nm; the maximum excitation wavelength of PI-DNA complex is 535 nm and the maximum emission wavelength is 615 nm. Draw a two-dispersion point graph using the relevant analysis software of the flow cytometer, with IF488 on the abscissa and PI 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 green fluorescence, and the scattered points are located in the second quadrant of the lower right. Late apoptotic cells and necrotic cells show red and green 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-IF488 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-IF488 has a green 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. If pancreatin is used during the operation, be sure to terminate the digestion with serum and remove residual pancreatin as much as possible.

 

4. After the adherent cell apoptosis is stimulated, some cells will float, and the cells in the cell culture supernatant will be collected at the same time, which will make the result more accurate.

 

5. Apoptosis is a continuous and dynamic process, and the fluorescent substance will be quenched, so the detection should be carried out as soon as possible after the reaction, and the whole process should also be protected from light as much as possible.

 

6. Early cell apoptosis detection is recommended to conduct preliminary experiments to optimize the corresponding steps.

 

7. 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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