Use of Far-Red Emitting DNA Dye DRAQ5 for Cell Cycle Analysis with Microplate Cytometry
Poster Sep 14, 2007
Sarah Payne, Paul Wylie, Roy Edward and Andrew Goulter
There is an increasing demand for multiplexing in high content assays to maximise data generation and allow correlation across multiple readouts.
Currently, the degree of multiplexing can be limited by the available reagents, with the majority of fluorescent probes optimised for excitation at 488 nm. The use of fluorescent probes with spectral profiles that overlap hinders their use in multiplexing assays. Another limitation is that many fluorescence detection systems excite and detect over a narrow wavelength range, making them incompatible with certain probes.
Laser-scanning fluorescence microplate cytometers, such as the Acumen® eX3 (TTP LabTech Ltd, Melbourn, UK), offer 405nm, 488nm and 633nm laser excitation in a single instrument. This technology is heavily used in oncology research including cell proliferation and cell cycle analysis using the DNA stains propidium iodide (488nm excitation) and Hoechst 34580 (405nm). Here, we describe the use of DRAQ5™ (633nm) on an Acumen eX3.
Use of DRAQ5 has become popular since it is a far-red fluorescent DNA dye that can be used in live and fixed cells in combination with other common fluorophores, especially GFP fusions and FITC-tags without spectral emission overlap. Thus DRAQ5 offers great potential for multiplexing DNA content analysis with immunodetection assays.
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