Cell Function FAQs

Annexin V kit can be used on adherent cells, with the following caveat:
1. In late stage of apoptosis, cells may not able wo maintain adhesion, so floating cells should be collected for the test.
2. The dissociation of adherent cells should be handled gently to avoid cell damage caused by human operation.
3. Do not digest for too long with trypsin, avoid the usage of the trypsin contain EDTA. If EDTA-containing trypsin must be used, wash the cells to remove EDTA as much as possible.

If there is a DAPI channel in the flow cytometry, the E-CK-A258 Annexin V-APC/DAPI Apoptosis Detection Kit will be suitable. If not, the E-CK-A218 Annexin V-APC/7-AAD Apoptosis Detection Kit is avaliable.

There is no such experience as verification yet. It is recommanded to take a pre-test if necessary.

It is not recommended to do so. Cryopreservation may lead to apoptosis and necrosis, resulting in inaccurate experimental results.

Annexin V-Elab Fluor® Red 780/DAPI Apoptosis Kit E-CK-A280 will be the best choice with minimun interference.
However, if there is no DAPI channel, Annexin V-Elab Fluor® Red 780/7-AAD Apoptosis Kit E-CK-A240 may also be avaliable.
Elab Fluor® Red 780 was detected by APC-CY7 channel. If there is neigher DAPI channel nor APC-CY7 channel, Annexin V-PE/7-AAD Apoptosis Kit E-CK-A216 could be a choice but with more interference.

Annexin V-APC/DAPI Apoptosis Kit E-CK-A258 is available, but requires a DAPI channel.

No, because Annexin V binds to phosphatidylserine (PS) dependent on Ca2+, which is not present in common PBS solutions, while Binding buffers contain Ca2+ to facilitate Annexin V binding to PS. Without Binding Buffer, Annexin V binding capacity will be reduced, resulting in significantly reduced positive signal or even no positive signal.

Annexin V assay is normally used for cell line samples, thus only debris need to be excluded by gating. Since there aremultiple types of cells in tissue samples, it depends on the purpose of experiment, whether it means to detect apoptosis of the whole tissue sample or some specific cells. Cells can be gated by FSC/SSC or some other biomarkers.
Besides, operation of tissue sample preparation may also effect the result, Inappropriate operation may also result in false positives due to sample handling.

The purpose of fixing cells is to preserve the original structure of tissues and cells as complete as possible, avoid degradation, autolysis, corruption, and deformation of tissues and cells, deactive various enzymes in cells and tissues to prevent various molecular degeneration and dissociation, and enable cell chemicals and enzymes to be accurately located. And in the future processing and production process will not change and damage. At the same time, fixation can also make each part of the cell easy to be stained, suitable for observation, long-term preservation and analysis.

Absolute ethanol is anhydrous ethanol which is 100%. It is not recommended to be replaced by 70% ethanol in order to match the best condition of assay.
If there is a sub-G1 spike, it can be detected. Due to nucleus concentration and DNA fragmentation, some genomic DNA fragments of apoptotic cells were lost during the staining process. Therefore, apoptotic cells showed significantlly weaker staining after PI staining than that of the normal cells, that is, the fluorescence intensity was less than 1. The so-called sub-G1 peak, or apoptotic cell peak, appeared on the flow detection result map.

Serum-free medium is necessary for cell cycle assays. Before the start of the experiment, cells should be cultured in serum-free medium for 12h to synchronize the cycle (pay attention to cell status, do not culture cells without serum for too long, or the cell state may be too poor to proliferate, end up with shrunk G2 phase or even sub-G1 apoptotic peak). Then normal medium was used for drug treatment, intervention and other experiments, and finally samples were collected for cell cycle detection.