PT  - JOURNAL ARTICLE
AU  - Edward, Roy
AU  - Dimmick, Ian
TI  - Compensation-free dead cell exclusion: multi-beam excitation of the far-red DNA binding viability dye DRAQ7. (TECH2P.873)
DP  - 2014 May 01
TA  - The Journal of Immunology
PG  - 135.4--135.4
VI  - 192
IP  - 1 Supplement
4099  - http://www.jimmunol.org/content/192/1_Supplement/135.4.short
4100  - http://www.jimmunol.org/content/192/1_Supplement/135.4.full
SO  - J. Immunol.2014 May 01; 192
AB  - Reliable exclusion of dead/damaged cells is often needed for immunological assays by flow cytometry and cell sorting. DRAQ7 is a far-red biomarker of cell viability developed for this purpose. This water-soluble molecule binds dsDNA with high affinity but is membrane impermeant, only labeling cells with compromised membranes (Akagi, J. et al., 2013 and Smith, P.J. et al., 2013). Multi-colour analysis is limited by the available spectrum and preference for bright fluors, increasing compensation issues. DRAQ7 aids panel design by its emission: not overlapping Brilliant Violet dyes, FITC or R-PE and minimally with APC. Due to its specificity and affinity it provides clear s:n in membrane-compromised cells. DRAQ7 is optimally excited at 599 / 644 nm, however it is usefully excited down to blue wavelengths. In principle, this, so called, multi-beam excitation means DRAQ7 occupies multiple fluorescence channels. Conversely, this actually enables selection of a unique population of dead cells; plotting two independently excited far-red channels against each other one can choose a suitable pairing that gives the best separation of a double-positive population for a chromophore panel. This double-positive population is defined as a dead-cell gate of events then entirely excluded from other channels (avoiding any compensation). This can be achieved during acquisition or post-acquisition. The technical principles of this simple approach and exemplar data will be presented.