生物制药

实验概要
Provides a rapid and convenient assay for apoptosis.

实验原理
Apoptosis is a carefully regulated process of cell death that occurs as a normal part of development. Inappropriately regulated apoptosis is implicated in disease states, such as Alzheimer’s disease and cancer. Apoptosis is distinguished from necrosis, or accidental cell death, by characteristic morphological and biochemical changes, including compaction and fragmentation of the nuclear chromatin, shrinkage of the cytoplasm, and loss of membrane asymmetry. In normal live cells, phosphatidyl serine (PS) is located on the cytoplasmic surface of the cell membrane. However, in apoptotic cells, PS is translocated from the inner to the outer leaflet of the plasma membrane, thus exposing PS to the external cellular environment. In leukocyte apoptosis, PS on the outer surface of the cell marks the cell for recognition and phagocytosis by macrophages. The human anticoagulant, annexin V, is a 35–36 kDa Ca2 -dependent phospholipid-binding protein that has a high affinity for PS. Annexin V labeled with a fluorophore or biotin can identify apoptotic cells by binding to PS exposed on the outer leaflet.

主要试剂
Alexa Fluor® 488 annexin V
Propidium iodide
PBS [Details：phosphate-buffered saline.]

实验步骤 细胞实验技术论坛 cuturl('http://bbs.bbioo.com/forum-138-1.html')
1. Induce apoptosis in cells using the desired method. Prepare a negative control by incubating cells in the absence of inducing agent.
2. Harvest the cells after the incubation period and wash in cold phosphate-buffered saline (PBS).
3. Prepare 1X annexin-binding buffer. For example, for ~10 assays, add 1 mL 5X annexin binding buffer (Component C) to 4 mL deionized water.
4. Prepare a 100 µg/mL working solution of PI by diluting 5 µL of the 1 mg/mL PI stock solution (Component B in 45 µL 1X annexin-binding buffer. Store the unused portion of this working solution for future experiments.
5. Re-centrifuge the washed cells (from step 2), discard the supernatant and resuspend the cells in 1X annexin-binding buffer. Determine the cell density and dilute in 1X annexin-binding buffer to ~1 × 106 cells/mL, preparing a sufficient volume to have 100 µL per assay.
6. Add 5 µL Alexa Fluor® 488 annexin V (Component A) and 1 µL 100 µg/mL PI working solution (prepared in step 4) to each 100 µL of cell suspension
7. Incubate the cells at room temperature for 15 minutes.
8. After the incubation period, add 400 µL 1X annexin-binding buffer, mix gently and keep the samples on ice.
9. As soon as possible, analyze the stained cells by flow cytometry, measuring the fluorescence emission at 530 nm (e.g., FL1) and >575 nm (e.g., FL3).