A novel assay to measure loss of plasma membrane asymmetry during apoptosis of adherent cells in culture
✍ Scribed by Manon van Engeland; Frans C. S. Ramaekers; Bert Schutte; Chris P. M. Reutelingsperger
- Publisher
- John Wiley and Sons
- Year
- 1996
- Tongue
- English
- Weight
- 856 KB
- Volume
- 24
- Category
- Article
- ISSN
- 0196-4763
No coin nor oath required. For personal study only.
✦ Synopsis
Early during the process of apoptosis, cells lose their phospholipid membrane asymmetry and expose phosphatidylserine (PS) at the cell s h c e while maintaiaing their plasma membrane integrity intact. This process can be monitored for suspended cell types by using annexin V-FITC, which is a Ca*+-dependent, phospholipid-binding protein with high afbity for PS, and flow cytometry. If adherent cell types are to be studied for t h i s apoptosisassociated phenomenon, then a problem is encountered, in that specxc membrane damage occurs during harvesting.
In this paper, a flow cytometric-based method is described that allows the measurement of loss of phospholipid asymmetry during apoptosis of ad-herent cells in culture. The method relies on the phospholipid binding property of biotinylated annexin V. Furthermore, the use of t h i s conjugate allows tricolor flow cytometric analysis of apoptosis. Employing the method to MR65 cells, which were initiated by olomoucine to enter apoptosis, it is shown that PS exposure occurs early after the onset of apoptosis and, at the prevalent time-resolution, that PS exposure is accompanied by loss of both cytokeratin and DNA. The annexinV+ cells appear as a characteristic sub-G, peak in the DNA histogram.