First, the detection principle
When cells undergo apoptosis, the permeability of their cell membranes also increases, but the extent is between normal cells and necrotic cells. Using this feature, the cell suspension to be tested is stained with fluorescein, and the fluorescence intensity in the cell suspension is measured by flow cytometry to distinguish normal cells, necrotic cells, and apoptotic cells.
Flow cytometry has the following characteristics
1. The number of cells detected is large, so it reflects the apoptotic state of the population cells.
2, many related analysis available
3. Combined with the analysis of the DNA content of the tested cells, the cell cycle in which the apoptotic cells are located can be determined. However, the method of the law also has a complicated production of specimens. Non-in situ detection is not easy to limit the identification of denatured cells.
Second, the detection method
1. Hoechs-PI double staining method for detecting morphology and cell membrane integrity
When the cells undergo apoptosis, the permeability of the cell membrane is also increased, but the degree is between normal cells and necrotic cells. With this feature, the cell suspension to be tested is stained with fluorescein, and the cells are measured by flow cytometry. The fluorescence intensity of the cells in the suspension distinguishes between normal cells, necrotic cells, and apoptotic cells.
Common Hoechs-PI staining method, normal cells have dye rejection, fluorescent staining is very shallow, apoptotic cells mainly ingest hoechs-PI dye, showing strong blue fluorescence, while necrotic cells mainly take up propidium iodide (PI). Red fluorescent.
2, DNA fragment in situ labeling
In situ end-detection of apoptotic cell DNA fragments refers to the use of fluorescein, digoxigenin or biotin-labeled deoxyuridine triphate (UDTP) and ends with the cell (or tissue) structure remaining unchanged. The technique of detecting the DNA cleavage point after the color reaction of the terminal deoxynucleotide transferase (TdT) phase reaction with the 3' hydroxyl (-OH) end of apoptotic cell lysis.
There are two kinds of DNA fragment in situ labeling methods.
(1) In situ nick-translation (ISNT) technique, proposed by Fehsel et al. in 1994, which uses DNA polymerase I to attach a labeled nucleotide to the 3'-OH end of the fragment.
(2) In situ end labelling technique (ISEL), which is the TUNEL method, was proposed by Wijsman et al. in 1993 to connect the labeled DUTP to the 3'-OH end using TdT. Studies have shown that the sensitivity of TUNEL is much higher than that of TUNEL, especially for the detection of early apoptosis. The reason may be that DNA is mostly double-stranded at the same time when apoptosis occurs, and single-strand breaks are rare. ISNT is a single-stranded repair reaction that relies on DNA polymerase I, so the positive rate is low. DNA cleavage precedes morphological changes and decreased DNA content during apoptosis. Therefore, the method has higher sensitivity than the above methods, but the dead cells also have DNA crack formation. Therefore, ring death can also be a TUNEL positive reaction.
3. Annexin V combined with PI method for detecting changes in cell membrane composition
(1) Detection principle
Phosphatidylserine (PS) located inside the cell membrane in the early stage of apoptosis migrates to the outside of the cell membrane. The phosphatidyl-binding protein V (Annexin V) is a calcium-dependent phospholipid-binding protein with a high affinity for PS. Therefore, Annexin V can be used as a needle to detect PS exposed on the surface of cell membranes. Therefore, using Annexin V with high affinity for PS, Annexin V was labeled with fluorescein, and apoptotic cells were detected by flow cytometry after double staining with apoptotic cells in combination with PI staining.
(2) Judgment of results
The normal living cells Annexin V and PI were all low-stained; the apoptotic cells Annexin V were highly stained and PI was low-stained; the necrotic cells Annexin V and PI were highly stained.
(3) Application value
When cells undergo apoptosis, PS exposure on the membrane occurs earlier than DNA fragmentation. Therefore, Annexin V combined detection of early apoptosis is more sensitive than TUNEL. Moreover, Annexin V combined PI staining method does not need to fix cells, and can avoid the excessive cell debris caused by PI staining and the loss of DNA fragments caused by TUNEL fixation. Therefore, the Annexin V combined method PI is more time-saving and more reliable, and is currently the most ideal method for detecting apoptosis.
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