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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

Figure 1.

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

Figure 2.


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

Figure 3.


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

Figure 4.

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

Figure 5.

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

Figure 6.

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~

Welcome to view and order Elabscience® Annexin V Apoptosis Detection Kit