핵 염색을 이용한 세포사멸 유도물질 스크리닝의 조건 비교

Comparison of Conditions for Cell Death-Inducing Agents Using a High Throughput-Compatible Nuclear Staining Assay

DAPI 염색을 이용하여 세포사멸 유도물질의 초고속스크리닝에 적합한 protocol을 제작하였다. Liquid handler에서 dispensing과 multilabel counter에서의 상대적인 fluorescent intensity를 측정하는 과정을 자동화한 protocol을 제작하였고, 이의 과정은 매우 효율적으로 가동되었음을 확인하였다. DAPI에 binding된 DNA의 intensity를 측정하는 조건검토에서 가장자리의 36개를 reading하지 않는 것이 edge effect를 줄일수 있는 것을 확인하였고, 0.1 sec reading으로 시간을 절약할 수 있었다. 이의 결과를 효과적으로 이용할 경우, 세포사멸 유도물질의 초고속스크리닝 또는 이에 적합한 스크리닝이 가능함을 확인하였다.

High throughput-drug screening plays a pivotal role for early stage of drug discovery process. In the course of assay development for screening of cell death-inducing agents, a protocol that is simple, time-saving, and high throughput-compatible was designed which was confirmed that the protocol can be worked by a HTS-compatible machine. In 96-well format, PC-3 cancer cells (1${\times}10^{4}$ cells/ml) were cultured for 24 hr. After 24 h-incubation with various medicinal plants extracts, the cells were then stained with DAPI for 30 min. The fluorescence intensity of the stained cells was measured semi-automatically with a multilabel counter. To test whether the present assay system effectively works, we screened about 850 medicinal plant extracts, and selected 1 positive crude extracts that contained cell death-inducing activity. These results suggest that the protocol is highly amenable to HTS implementation for a cell death-inducing agent(s).