High Throughput Cell Cycle Analysis using Microplate Cytometry
Poster Feb 01, 2007
Tristan Cope and Wayne Bowen
The cell cycle is a target for many anti-cancer drugs and the ability to monitor the effects of such agents on the cell cycle is an important part of the drug development process. Standard methods measure changes in DNA content by staining the nuclei of fixed cells with propidium iodide. The cells are then sorted by flow cytometry into G1, S and G2/M populations according to fluorescent intensity. The main disadvantages of this technique are low throughput, use of large number of cells and an inability to analyse adherent cell lines in situ.
To improve screening efficiency, we have developed a cell cycle analysis method that uses an Acumen Explorer fluorescence microplate cytometer to measure the DNA content of propidium iodide stained fixed cells in microplates. We demonstrate that paclitaxel and vinblastine arrested CHO cells in the expected phase of the cell cycle.
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