Monitoring apoptosis with fluorescent Zn2+-indicators.
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.
Kimura
Eiichi
E
Faculty of Integrated Arts and Sciences, Hiroshima University, 1-7-1 Kagamiyama, Higashi-Hiroshima 739-8521, Japan. ekimura@hiroshima-u.ac.jp
Takasawa
Ryoko
R
Tanuma
Sei-ichi
S
Aoki
Shin
S
D017304:Annexin A5 / Q000737:chemistry / Q000378:metabolism; D017209:Apoptosis / Q000502:physiology*; D002232:Carbocyanines / Q000737:chemistry / Q000378:metabolism; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D053938:DNA Fragmentation; D003619:Dansyl Compounds / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells / Q000737:chemistry / Q000473:pathology / Q000502:physiology; D006573:Heterocyclic Compounds, 1-Ring / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D007364:Intercalating Agents / Q000737:chemistry / Q000378:metabolism; D007476:Ionophores / Q000737:chemistry / Q000378:metabolism; D009336:Necrosis; D011419:Propidium / Q000737:chemistry / Q000378:metabolism; D015363:Quinolones / Q000737:chemistry / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods; D014105:Tosyl Compounds / Q000737:chemistry / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
D017304:Annexin A5; D002232:Carbocyanines; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D006573:Heterocyclic Compounds, 1-Ring; D007202:Indicators and Reagents; D007364:Intercalating Agents; D007476:Ionophores; D015363:Quinolones; D014105:Tosyl Compounds; C089187:cyanine dye 3; D011419:Propidium; D004247:DNA; D015032:Zinc; C084731:zinquin