Annexin V is used as a probe to detect cells that have expressed phosphatidylserine (PS) on the cell surface, an event found in apoptosis as well as other forms of cell death. The Annexin V affinity assay typically uses a conjugate of annexin V and a fluorescent or enzymatic label, biotin or other tags, or a radioelement, in a suitable buffer (Annexin V binding to PS is Ca²⁺ dependent). The assay combines Annexin V staining of PS membrane events with the staining of the cell nucleus with PI or AAD-7 to distinguish living cells from dead cells. Annexin V apoptosis detection is based on the observation that soon after initiating apoptosis, cells translocate the membrane phosphatidylserine (PS) from the inner (cytoplasmic-facing) leaflet of the plasma membrane to the cell surface. Once on the cell surface, PS can be easily detected by staining with a fluorescent conjugate of Annexin V, a protein that has a high affinity for PS. Detection can be analyzed by flow cytometry or by fluorescence microscopy.

This kit contains:

• Annexin V (APC): 500 µl
• 7-AAD Viability Staining Solution: 500 µl
• Positive Control: 5 ml
• 10X Binding Buffer

Target	Annexin V
Reactivity	General (All species)
Conjugation	APC
Excitation/Emission	651/660
Laser Line	647
Form	Liquid
Storage	10X Binding Buffer: Store at 4 °C for short-term storage. For long-term storage, aliquot and store at -20 °C. All other reagents: Store undiluted at 4 °C. Avoid exposure to light. Do not freeze.
Directions for use	Staining Protocol Dilute the 10X Binding Buffer solution to 1X Working Binding Buffer solution with distilled water. Harvest cells (about 1 × 105 cells per test), then wash once with cold PBS. Remove the PBS from the cell pellet. Wash again with cold 1X Working Binding Buffer, then centrifuge at 300 × g for 10 min at room temperature. Remove the Binding Buffer from the cell pellet. Resuspend cells in cold 1X Working Binding Buffer to a concentration of 1 × 106 cells/ml. Add 100 µl of cells (1 × 105 cells) to each appropriate tube. Add 5 µl of Annexin V-FITC to the appropriate tubes. Gently vortex each tube and incubate for 10 minutes at room temperature in dark. Add 5 µl of PI or 7-AAD solution and incubate for 5 min at room temperature in dark. Wash cells once in PBS, then resuspend in PBS. Analyze by flow cytometry within 4 hours.
Availability	Shipped within 5-10 working days.
Note	This product is for research use only.

Research Articles on Annexin V (APC) / 7-AAD Apoptosis Detection Kit

Isotype Control 同型对照 同型对照（Isotype Control），使用与一抗相同种属来源、相同亚型、相同剂量和相同的免疫球蛋白及亚型的免疫球蛋白，用于消除由于抗体非特异性结合到细胞表面而产生的背景染色。 同型对照 同型对照的主要目的是确定一抗的结合是特异性的，而不是非特异性的Fc受体或与其他蛋白的相互作用。还可以用来竞争性的结合抗体，与功能阻断抗体发挥同样的功能。 同型对照要与一抗的来源，Ig分型和标记完全一致。 如果一抗是多抗，可以用an normal serum（与一抗相同的正常血清） （must be the same species as primary antibody）。This control is easy to achieve and can be used routinely in immunohistochemical staining.这个可以咨询试剂商。同型对照为免疫荧光标记中的阴性对照。由于荧光标记单抗的组来源不同，应选用相同来源的未标记单抗作为同型对照来 调整背景染色。举个例子：比如检测一抗为单抗的mouse anti rat CD11b，clone OX-42 purified IgG，那么它的isotype 是mouse IgG2a， 所以可以用purified（纯化的） mouse IgG2a来做OX-42的同型对照（Isotype Control）。一般的的生物技术公司和国内的代理都有出售。 同型对照：是指与MoAb相同的、未免疫小鼠的免疫球蛋白亚类，若使用直接免疫荧光染色法，同型对照也应标记荧光色素，如IgG1 FITC、IgG2a、PE等。主要考虑了细胞的自发荧光、FC受体介导的抗体结合和非特异性抗体结合等影响因素。此外，同型对照与MoAb所标记的荧光 色素、浓度、F：P比值（标记的荧光色素与免疫球蛋白分子的比值）应该相同为最佳，这对准确设定阴性与阳性细胞的界标有重要意义，切忌使用与MoAb不相 匹配的同型对照，最好为同一实验室、采用相同工艺或方法制备（如同一品牌）的产品。