Cell Function
The Data Still Doesn't Match? Correlation Analysis of Annexin V & CCK-8 Results
Source: Elabscience®Published: Jan 16,2024
Recently, we published the article: The data doesn't match? Correlation analysis of cell cycle &CCK-8 results to explain the CCK 8 and the connection between the cell cycle, there is another question: The results of cell cycle are consistent with CCK-8, but the results of Annexin V apoptosis are still inconsistent with CCK-8.
Don't worry, this article will take you to look at the relationship between Annexin V and CCK-8.
The solution is still the same. Before make clear the relationship between the Annexin V and CCK 8, must first understand their true detection indicators.
CCK-8
CCK-8 detects the total dehydrogenase activity in the sample. As long as the dehydrogenase activity in the sample to be detected changes, the OD detected by CCK-8 will change accordingly. At the same time, because the CCK-8 detect result is ultimately reflected as the OD value, the absorbance change caused by other reasons may also cause the OD value to change. More CCK 8 abnormal causes and elimination method in detail in the previous articles, interested reader can click to view (The data doesn't match? Correlation analysis of cell cycle &CCK-8 results) to learn more about it.
Annexin V
Annexin V detects phosphatidylserine (PS), which is exposed to the apoptosis cell surface. Normally, the nuclear dye used with Annexin V is able to enter cells with altered membrane permeability and bind to the nucleic acids.

According to the experimental principle, as long as the cell membrane has phosphatidylserine (PS), it can bind to Annexin V; As long as the nuclear dye can enter the cell and the cell has the nucleus, it can be bound by the nuclear dye. Therefore, before analyzing the correlation between CCK-8 and Annexin V results, it is important to rule out various abnormalities in Annexin V assay:
(1) There is a large amount of phosphatidylserine (PS) on the outer surface of normal platelets, so Annexin V cannot be used to detect platelet apoptosis;
(2) Mature mammalian red blood cells and platelets have no nuclei, so nuclear dyes cannot bind to these cells;
(3) Some drugs may destroy phosphatidylserine (PS) or DNA, which may cause the assay reagents to not bind correctly;
(4) When adherent cells undergo apoptosis, the late regulated cells will float up, and the absence of supernatant may lead to the loss of late regulated population.
(5) The cell seeding density is too high, which causes the cells to be "crowded to death";
(6) For more information on Annexin V sample preparation and abnormal detection, please go to read the notes of Annexin V Apoptosis assay.
After resolving the above abnormalities, we can formally analyze the correlation between CCK-8 and Annexin V results:
CCK-8 |
Annexin V |
Reference Reason |
Inhibiting increment |
Increased apoptosis rate |
After cell treatment, it is common to induce apoptosis, thereby inhibiting cell proliferation |
Decrease in apoptosis rate |
After cell treatment, cells grow at a slower rate or go dormant |
|
No change in apoptosis rate |
a) Cells are not treated with sufficient intensity to cause apoptosis b) The inhibitory effect is not caused by the apoptotic pathway |
|
Promotion of proliferation |
Increased apoptosis rate |
It is commonly seen in inflammatory reactions, where immune cells proliferate rapidly and die in response to inflammatory reactions |
Decreased apoptosis rate |
Therapeutic protective drugs, which can optimize the cell state, promote proliferation, and reduce apoptosis |
|
No change in apoptosis rate |
After cell treatment, the proliferation is changed, but the cell state does not change significantly, which is normal situation. |
This is the end of Flow Cytometry experimental knowledge sharing in this issue, hoping to help you complete the experiment more smoothly.
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