Cell Cycle Analysis Using Microplate Cytometry: A Comparison of Laser and Dye Combinations
Poster Mar 14, 2006
Tristan Cope, Christopher Lupton, Paul Wylie, Jeff Hung and Wayne Bowen
AbstractMany anticancer agents and carcinogens are DNA damaging chemicals and exposure to such chemicals results in the deregulation of cell cycle progression. The cell cycle is thus a target in oncology research, making the ability to monitor the effects of such agents on the cell cycle an important part of the drug development process. Traditionally, cell cycle analysis has been performed using flow cytometry.
For improved screening capability, we have developed a cell cycle analysis method using an Acumen Explorer fluorescence microplate cytometer, capable of reading an entire 384 well microplate in about 10 minutes. The method can perform such analyses on fixed cells in situ, markedly simplifying sample preparation. Here, we compare the performance of different combinations of laser excitation and fluorescent DNA labelling dyes on an Acumen Explorer microplate cytometer.
We utilized paired synthetic crRNAs coupled with our synthetic tracrRNA in cells transduced with lentiviral Cas9 to perform a functional knockout on hsa-miR-221. This three-part system (crRNA, tracrRNA and Cas9) has demonstrated efficient gene editing when used with only one guide RNA, but the goal was to use two crRNAs to remove the entire stem-loop.READ MORE