Monitoring Apoptosis with Fluorescent Zn2+-Indicators
Eiichi Kimura1*,
Ryoko Takasawa2,
Sei-ichi Tanuma2,3, and
Shin Aoki3*
1Faculty of Integrated Arts and Sciences, Hiroshima University, 1-7-1 Kagamiyama, Higashi-Hiroshima 739-8521, Japan.
2Genome and Drug Research Center, Tokyo University of Science, 2641 Yamazaki, Noda 278-8510, Japan.
3Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda 278-8510, Japan.
Abstract:
Apoptosis, a mechanism of programmed cell death that removes superfluous and harmful cells, is important both during development and in tissue homeostasis. Although Zn2+ is believed to be critical in apoptosis, the precise details of its role have yet to be elucidated. The macrocyclic Zn2+ ligand dansylamidoethylcyclen [L1•(HCl)4•(H2O)2], which is found primarily in a diprotonated form (H2L1), is cell-permeable and forms a strongly fluorescent 1:1 Zn2+ complex when Zn2+ entry into cells is facilitated by the Zn2+ ionophore pyrithione. H2L1 can be used to readily identify HeLa cells undergoing the early stages of etoposide-induced apoptosis because of the increased level of free Zn2+ that occurs at this time. The selectivity of H2L1 for the detection of apoptotic cells was verified by a conventional probe for apoptosis, annexin V-Cy3. Here, we describe methods for detecting apoptotic cells with H2L1 and for comparing detection of apoptosis with H2L1 to detection with annexin V-Cy3 and Zinquin.
*Corresponding authors. E-mail: ekimura{at}hiroshima-u.ac.jp, shinaoki{at}rs.noda.tus.ac.jp
