Multicolor Detection of CD45 on Human Monocytes with Quantum Dots in a High-Throughput Format
Poster Feb 20, 2006
Paul Wylie, John Budd and Marcel Bruchez
AbstractMulticolor quantum dot probes emitting from 525 to 705 nm can be effectively excited with a single 488 nm laser, resulting in signals equal to the best organic dyes, but that are cleanly discriminated with simple color filters. Using a plate based 4-color laser scanning system with a single 488 or 405 nm laser for excitation, we have detected the binding of quantum dot conjugates to CD45 on human monocytes.
The colors were easily discriminated with little or no spill-over in the detector for adjacent colors, allowing very clean discrimination of up to four individually labelled cells in a mixed population in a single well, as well as clear detection and quantitation of relative signals on cells labelled with multiple quantum dots. The lack of color overlap in these materials enables the detection of multiple cellular parameters in a high-throughput cell-analysis system, increasing the information density of a screen.
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