Cell Function
Analysis and Solution of Common Problems in Annexin V Detection
Source: Elabscience®Published: Jan 16,2024
What should we do when there are abnormal experimental results in the Annexin V apoptosis experiment?
We need to take failed experimental data seriously, analyze and summarize the possible reasons for experimental failure, so as to conduct targeted experiments again and obtain ideal experimental results.
This article provides an analysis of common problems in the Annexin V experiment, and through practical cases, helps you gain a deeper understanding of the Annexin V experiment.
Problem 1: The late apoptotic cells in the treatment group are divided into two groups, lacking early apoptosis

Possible reasons: The cell processing conditions are too intense, such as high drug concentration, excessive use of organic solvents to dissolve drugs, and extreme conditions (boiling water, etc.) that treat cells, resulting in rapid cell death, similar to the effect of cell cycle detection where cells are fixed, lacking the process of apoptosis.
Solution: Gently treat cells, such as reducing drug concentration; Control the amount of organic solvents (such as DMSO) below 5 ‰.
Problem 2: Lack of early apoptotic cells, accompanied by a large number of necrotic or naked nuclear cells

Problem 3: No positive signal in nuclear staining (PI/7-AAD/DAPI)

Possible reasons |
Solution |
Forgot to add nuclear dyes |
Re experiment, pay attention to adding nuclear dyes |
Reagent failure due to improper storage, such as 7-AAD not being stored at -20℃ |
Repurchase reagents and pay attention to the storage conditions of the reagents |
The cells did not undergo apoptosis |
Readjust the cell processing conditions by observing whether the cells are apoptotic under a microscope |
The threshold is set too high, and the apoptosis signal is not collected |
Adjust instrument settings and lower the threshold |
The cells in the supernatant of the adherent cell culture medium were not collected |
Re experiment, pay attention to collecting cells from the supernatant |
Problem 4: Unclear clustering

Possible reasons |
Solution |
Cells have spontaneous fluorescence |
Replace reagent kits with other fluorescent |
Excessive cell apoptosis leads to insufficient dyes |
Increase dye usage |
The cell state is poor, and all cells have a certain degree of PS eversion |
Both cultivation and experimental processes require gentle treatment of cells |
Problem 5: There is a fluorescence signal in the blank group

Possible reasons |
Solution |
The Flow Cytometer was not cleaned thoroughly |
Thoroughly clean the Flow Cytometer |
Interference from fluorescent substances (such as doxorubicin, tetracycline, cells transfected with fluorescent plasmids, etc.) |
Replace with other fluorescent labeled reagent kits, or culture cells with other substances |
Background fluorescence of apoptosis or necrosis |
The blank group should select normal cells in good condition. If the cell state is too poor to recover or there is contamination, the cells should be replaced in a timely manner |
Cellular impure |
Problem 6: Normal cells exhibit a significant amount of apoptosis

Possible reasons |
Solution |
Poor cellular status |
Adjust cell state and culture cells again |
Rough handling during experimental operations or excessive digestion of cells |
Treat cells gently |
Long incubation time and prolonged exposure to abnormal growth environments can lead to cell apoptosis |
Control the time of the experiment well. If there are too many samples, the experiment can be conducted in batches |
Dilution issue with Binding Buffer: Buffer was incorrectly diluted,, abnormal solution osmotic pressure leading to cell apoptosis |
Follow the instructions for operation |
The above is an analysis and solutions of common problems in the Annexin V experiment, hoping to be helpful for everyone's experiment~
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