Assay of Cell Apoptosis (Annexin-V-FITC)

Experimental Principle

Apoptosis or programmed cell death (PCD) is one of the cellular life phenomena that plays an important role in embryonic development, tissue repair and clearance of self-reactive T lymphocytes in the organism. The deregulation of apoptosis can lead to the development of various clinical diseases. For example, Alzheimer's disease is associated with excessive apoptosis in neuronal cells, and autoimmune diseases and tumors are associated with inhibition of apoptosis. Apoptosis is distinct from necrosis in that it has a range of morphological and biochemical cellular changes, including the appearance of chromatin condensation, DNA degradation, and apoptotic vesicle formation. In normal living cells, phosphotidylserine (PS) is located on the inner side of the cell membrane, but in apoptotic cells, PS flips from the inner side of the cell membrane to the surface of the cell membrane and is exposed to the extracellular environment. Annexin-V (membrane-linked protein-V) is a Ca2+-dependent phospholipid-binding protein with a molecular weight of 35-36 KD that binds to PS with high affinity binding. Therefore, Annexin-V is labeled with fluorescein (e.g., FITC, PE) or biotin, and the labeled Annexin-V is used as a probe to detect the onset of apoptosis using flow cytometry or fluorescence microscopy.

Main Reagents

Annexin V-FITC (membrane-linked protein-V-FITC) 500 μl/250 μl 20 μg/ml
Labeling Buffer (Binding Buffer) 40/20 ml
PBS (1×) 60/30 ml

Main Equipment

20 μl, 200 μl, and 1000 μl pipettes and tips
Microcentrifuge tubes
Adjustable speed centrifuge
Slides (for fluorescence microscopy)
Coverslips (for fluorescence microscopy)
Deionized water
Ice cubes
Flow cytometer or fluorescence microscope

Experimental Materials

Mice

Experimental Steps

Preparation of apoptotic cells: mice were injected intraperitoneally with dexamethasone 25 mg/kg body weight, and the thymus was dissected and taken after 10 h. Thymus cells were isolated. Wash twice with PBS, adjust the concentration of cells to be tested to 2 × 106/ml, and set aside.
200 μl of cell suspension was added to 1 ml of cold PBS, and the cells were suspended by gentle shaking and centrifuged at 1000 rpm for 10 min at 4°C.
Repeat step 2 twice.
Resuspend the cells in 200 μl of labeling buffer.
Add 10 μl Annexin V-FITC, mix gently, and react for 15 min at room temperature or 30 min at 4°C, protected from light.
Add 300 μl of labeling buffer and assay immediately on the machine (flow cytometry).

Cautions

The whole operation should be as gentle as possible, do not blow the cells with force, and operate at 4℃ as much as possible.
After the reaction, the cells should be detected as soon as possible, because apoptosis is a dynamic process and the fluorescence intensity starts to decay after one hour of reaction.
Annexin V-FITC is a photosensitive substance, so be careful to avoid light during operation.

* For research use only. Not intended for any clinical use.

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Assay of Cell Apoptosis (Annexin-V-FITC)

Experimental Principle

Main Reagents

Main Equipment

Experimental Materials

Experimental Steps

Cautions

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