Annexin A6 propidium iodide for cell cycle specificity of apoptosis detection

Apoptosis is an important aspect of cell biology, apoptosis detection method based on flow cytometry (FCM), a variety of terminal deoxynucleotidyl transferase-mediated DNA strand nick labeling method (TdT) is the only on cell proliferation and apoptosis of the cell cycle-specific method for simultaneous analysis of mark steps of the method prone to false negative results, and not sensitive to early apoptotic cells. Conventional fluorescein isothiocyanate (FITC) labeled ANXA6 Act (FITC2Annexin A6) to establish on the basis of the phenomenon of apoptotic cell membrane eversion, therefore, provides a cycle-specific apoptosis detection possibilities.Known apoptosis, membrane eversion phenomenon, the inside of the plasma membrane phosphatidylserine is exposed on the outer surface of the membrane of apoptotic cells typical membrane structural changes early in apoptosis already occurred. Use by Annexin the A6 with the negatively charged phosphatidylserine, apoptotic cell membrane PI refuse to different characteristics of the dye ability to establish a routine by Annexin A6 apoptosis assay, can be effective for quantitative detection of apoptosis in different times , and can distinguish between necrotic cells. On the basis of this principle, if the PI intake capacity increase of apoptotic cells, staining the nuclear DNA, while not affecting by Annexin A6 phosphatidylserine binding capacity, we can establish a both quantitative apoptotic cells , but also apoptosis of the cell cycle specific analysis.

The study found that can effectively prevent the fixed cells with methanol-free formaldehyde solution by Annexin A6 dissociation, the formaldehyde concentration of 1% is better. Living cells after formaldehyde pre-fixed, and then digitonin on cell membrane permeability processing will not affect the membrane markers.Concentration of 500 mg / L digitonin-treated cells, can effectively increase the ability of the PI membrane penetration and staining of nuclear DNA, and does not affect the combination of phosphatidylserine by Annexin the A6, it will not lead to intracellular small molecular weight DNA leakage. Conventional rupture of membranes reagent of Triton the X2100 will be lead by ANXA6 dissociation. By Annexin A6 and phosphatidyl serine combination has of Ca2 +-dependent characteristics of the Ca2 + concentration (5mmol / L), shall be maintained in the process of cell marker, otherwise easily lead by Annexin the A6 dissociation of FITC The fluorescence intensity decreased apoptosis detection.To deal with HL260 cells by camptothecin and TPCK, TPCK inhibit the degradation of DNA between nucleosomes, but does not prevent the DNA strands split, the DNA chain only fracture the formation of a molecular weight of the large fragment of more than 50000 cells in a withered death in the early stages. DNA strand gap is relatively small, the TdT-law can not distinguish between apoptotic cells; the other hand, TPCK does not affect the apoptotic cell membrane eversion, with the PA method can still be effectively detected.

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