Use of a fluorescence lifetime imaging microscope in an apoptosis assay of Ewing's sarcoma cells with a vital fluorescent probe.
A fluorescence lifetime imaging microscope (FLIM) was applied to study early-stage apoptotic cells stained with a SYTO13 dye. The fluorescence lifetime of SYTO13 in healthy cells was 3.8+/-0.3ns but was reduced to 2.4+/-0.4 and 1.9+/-0.2ns after a 3-h period of incubation with SYTO13 when doxorubicin, a known inducer of apoptosis, was added to human Ewing's family tumor cells at final concentrations of 250 and 500nM, respectively, in a dose-dependent experiment. On the other hand, in a time-dependent experiment, the fluorescence lifetime decreased to 2.5+/-0.5 and 1.7+/-0.4ns at a doxorubicin concentration of 750nM after 2 and 4h, respectively. A possible explanation for these results is self-quenching induced by a change in interprobe distance that arises from the condensation of DNA during apoptosis. In this study, the FLIM system was employed to investigate early-stage apoptosis that involves only small morphological changes, suggesting the potential advantage of this method for evaluating small biological effects in living cells.
Li
Xu
X
Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Nishi-ku, Fukuoka 819-0395, Japan.
Uchimura
Tomohiro
T
Kawanabe
Satoshi
S
Imasaka
Totaro
T
D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000494:pharmacology; D005456:Fluorescent Dyes; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D009930:Organic Chemicals; D012512:Sarcoma, Ewing / Q000473:pathology*
D005456:Fluorescent Dyes; D009930:Organic Chemicals; C461159:SYTO 13; D004317:Doxorubicin