Resources Technical Documents DRAQ5 and BD PATHWAY 855
Technical documents description.....
Scientists at the CNIO in Madrid have developed an exciting new technology allowing the specific monitoring of compounds which interfere with pathways in tumour cells. As their model system they used the akt pathway. Briefly, Il-3 dependent Ba/F3 cells were stably transfected with EYFP or ECFP and then rendered respectively akt-dependent or PI3K/akt independent (by generating STAT5 over-expression). In both cases the modified cells were Il-3 independent.
The isogenic mixture of YFP/akt and CFP/STAT5 cells was then challenged with a series of compounds with known activity. A decrease in the ratio of yellow and blue cells indicated inhibition of the akt pathway, making cells Il-3 dependent again leading to apoptosis. Compounds with generalised cytotoxicity were indicated by reduction of the numbers of DRAQ5-positive nuclei over the 12h time-course of the experiment. Examples of the images generated are shown in the 6 panels below.
DRAQ5 (shown as red) was the perfect choice as nuclear counterstain in this procedure since it efficiently labels live cells, demarks the nucleus and has no spectral emission overlap with either CFP (blue) or YFP (yellow).
REFERENCE: Rosado A, Zanella F, Garcia B, Carnero A, Link W (2008) A Dual-Color Fluorescence-Based Platform to Identify Selective Inhibitors of Akt Signaling.
PLoS ONE 3(3): e1823. doi:10.1371/journal.pone.0001823 (link to the article here ).
Biostatus is very grateful to Dr Wolfgang Link, CNIO, Madrid for use of the images.
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