流式細胞術檢測細胞凋亡

實驗概要

Provides a rapid and convenient assay for apoptosis.

實驗原理

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.

主要試劑

Alexa Fluor? 488 annexin V

Propidium iodide

PBS [Details：phosphate-buffered saline.]

實驗步驟

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).

注意事項

Propidium iodide is a potential mutagen, use appropriate precautions when handling this reagent.