Analysis of common problems in Annexin V apoptosis detection

Analysis of common problems:

During the experiment, Annexin V was not stained or the positive rate was low.
• Determine if the inducer in the experiment produces apoptosis. This can be ruled out by setting a positive drug control for the exact apoptosis-inducing effect.
• Implanted cells are not properly digested. The combination of Annexin V and PS requires Ca2+ ions. The binding solution contains Ca2+. The digestion of EDTA will affect the staining. It is recommended to use EDTA-free trypsin or wash it with PBS after digestion.
• After washing the cells with cold PBS, the residual liquid should be aspirated as much as possible. Excessive residual PBS contains phosphate, which will form calcium phosphate precipitate and affect the staining of Annexin V.
• The Annexin V combined liquid bottle cap should be tightly sealed. After prolonged exposure to air, the formation of CaCO3 after the CO2 in the air enters will precipitate, thus reducing the free Ca2+, resulting in poor experimental results.
• Contaminant other buffers. If the Tip head is not replaced after PBS is taken up, it will lead to a decrease in free Ca 2+ , resulting in poor experimental results.
• Loss of positive cells. If it is adherent cells, the cells floating after drug induction should be collected and combined. This part of the cells is often apoptotic-positive cells, and discarding will result in a low positive result.

The positive rate was high during the experiment.
• The cells themselves are too weak. In the experiment, it was found that the percentage of cells with AnnexinV+/PI+ double positive after staining of control cells without induction of apoptosis was too high, which may be caused by the poor viability of the cells themselves. Cell viability is recommended by trypan blue staining. Trypan blue exclusion should be greater than 95%. If the vitality is low, it is recommended to resuscitate the cells. Usually, the cells that have just recovered should be passaged at least 3 times before the experiment can be carried out.
• Improper cell manipulation. Repeated vigorous blows during the digestive process of adherent cells lead to destruction of the cell membrane, resulting in a high false positive.
• Apoptosis induction is too long. In general, early apoptosis can occur after several hours of induction, so it usually does not need to be processed for more than 48 hours. Excessive induction time will deplete nutrients, resulting in poor cell status and high false positive results.

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