Pacific Blue™ Annexin V/SYTOX™ AADvanced™ Apoptosis Kit, for flow cytometry - FAQs

使用胰蛋白酶消化或机械刮擦细胞暂时破坏质膜，使得膜联蛋白V结合到细胞膜胞内面上的磷脂酰丝氨酸，导致假阳性染色。胰蛋白酶消化/机械刮擦后，让细胞在最佳的细胞培养条件和培养基中复苏约30分钟，从而在染色前恢复细胞膜完整性。对于轻微粘连细胞系，例如HeLa和NIH 3T3，还可使用无酶处理方法，例如使用Gibco 细胞溶解缓冲液（货号13151014）。

膜联蛋白V染色一般不用于成像实验；而是流式细胞仪分析的最佳试剂。所有的细胞染色程度都会相近，因此很难从暗的非凋亡细胞中分辨出相对明亮的膜联蛋白V染色的细胞。使用我们的CellEvent Caspase 3/7 或Image-iT LIVE Caspase检测试剂盒检测caspase酶活化，这是用于成像实验检测凋亡的最佳方法。

先用胰蛋白酶消化，用膜联蛋白V偶联染料染色之前，在合适的细胞培养条件和培养基中复苏约30分钟。用胰蛋白酶消化或机械刮擦细胞暂时打乱了质膜，使得膜联蛋白V连接到细胞膜的胞内面上的磷脂酰丝氨酸，因此导致假阳性染色。对于轻微粘连细胞系例如HeLa和NIH 3T3，您可以使用低强度（无酶）的解离产品如Gibco Cell Dissociation Buffer（货号13151014）。

膜联蛋白V染色分析最好在是活细胞上进行。如果您需要固定您的细胞并用于分析，用3.7%甲醛在含有钙和镁的PBS溶液中固定可以维持连接。透化作用后信号不会保留，因此膜联蛋白V染色无法与内部抗体标记兼容。 

It has been done, but we don‘t recommend it. Both healthy cells and apoptotic cells possess phosphatidylserine on the cell surface, which can be detected with Annexin V, but apoptotic cells have significantly more of it. You can easily tell the difference between these two populations with flow cytometry, because flow cytometers are more sensitive and have a higher throughput. But with a microscope, you cannot always tell the difference, especially for adherent cells. Instead, for microscopy, we recommend a different technique, such as detecting caspases with CellEvent Caspase Detection Reagents.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Trypsinization or mechanical scraping of cells temporarily disrupts the plasma membrane, allowing annexin V to bind phosphatidylserine on the cytoplasmic surface of the cell membrane and thus leading to false positive staining. Allow the cells to recover for about 30 minutes in optimal cell culture conditions and medium after trypsinizing/scraping so that they can recover their membrane integrity before staining. For lightly adherent cell lines, such as HeLa and NIH 3T3, another option is to use non-enzyme treatments like Gibco Cell Dissociation Buffer (Cat. No. 13151014).

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Annexin V staining is not typically used in imaging experiments; it is a better reagent for flow cytometry analysis. All cells will stain to some extent, so it can be difficult to distinguish a relatively bright annexin V-stained cell from a dimmer non-apoptotic cell. Caspase activation, detected using our CellEvent Caspase 3/7 or Image-iT LIVE Caspase detection kits, is a better method for detecting apoptosis in an imaging assay.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Trypsinize first and then allow the cells to recover about 30 minutes in optimal cell culture conditions and medium before staining with annexin V conjugates. Trypsinization or mechanical scraping of cells temporarily disrupts the plasma membrane, allowing for annexin V to bind phosphatidylserine on the cytoplasmic surface of the cell membrane and thus leading to false positive staining. For lightly adherent cell lines such as HeLa and NIH 3T3, you could use a less harsh (non-enzymatic) dissociation product like Gibco Cell Dissociation Buffer (Cat. No. 13151014).

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Annexin V staining is best analyzed on live cells. If you need to fix your cells for analysis, then fix in 3.7% formaldehyde in PBS containing calcium and magnesium to maintain binding during fixation. The signal will not be retained after permeabilization, thus annexin V staining is not compatible with internal antibody labeling.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.