Quantification of apoptotic cells with fluorescein isothiocyanate-labeled annexin V in chinese hamster ovary cell cultures treated with cisplatin

✍ Scribed by Antonius W. M. Boersma; Kees Nooter; Robert G. Oostrum; Gerrit Stoter

• Publisher: John Wiley and Sons
• Year: 1996
• Tongue: English
• Weight: 740 KB
• Volume: 24
• Category: Article
• ISSN: 0196-4763
• DOI: 10.1002/(sici)1097-0320(19960601)24:2<123::aid-cyto4>3.0.co;2-k

✦ Synopsis

Plasma membrane binding of annexin V was used to detect and quantitate apoptotic cells induced by cytotoxic drug treatment in epithelial cell cultures. Chinese hamster ovary (CHO) cells were incubated for 2 h with the ID90 concentration of Cisplatin (20 pM), and 24,48,72, and 96 h later the unfixed cells were stainedwith fluorescein isothiocyanate (F1TC)- conjugated annexin V. The fluorescence signal was quantitated by flow cytometry (FCM). During the early phase of the apoptotic response, the annexin V-binding frequency histograms showed two separate cell populations, a dimly and a brightly fluorescent one. At t = 96 h after drug incubation, when the process of apoptosis was completed, only the brightly fluorescent population was present. A doseeffect relationship could be established between the Cisplatin concentration used in the 2 h incubation and the binding of annexin V on the cell membrane, as estimated by FITC fluorescence. The dimly and brightly fluorescent populations were sorted on the basis of annexin V binding, and assayed for 1) DNA