Monday, 22 August 2016

Improved, simplified FACS stem cell analysis strategies with DRAQ5

Researchers at UC-Merced in the lab of Nestor Oviedo recently published in the journal Regeneration a series of new and improved methods for the flow cytometric analysis of stem cells.  Their particular interest is the use of the planarians (flatworm) as model organisms. 
The authors describe improved flow cytometry analysis and FACS sorting protocols tested in the context of RNA interference (RNAi), with gamma irradiation or other cell stress. 
A key component of the improvements in a number of methods is use of the far-red vital DNA probe DRAQ5 alongside Calcein AM to give a clear indication of all nucleated cells and then those that are metabolically competent and have intact plasma membranes, respectively.  These two reagents have conveniently well-separated spectral properties making them ideally suited to use together.

DRAQ5 labels the DNA of all nucleated cells whether they are intact, damaged or dead. The stoichiometric labelling allows differentiation between cells in the phases of cell cycle, hypoploidy, hyperploidy and sub-G1 (suggesting apoptotic DNA loss).  Due to its cell permeance this means that DRAQ5 reports this information with minimised sample preparation.  Further, one can differentiate between debris and also doublets.  Full info on DRAQ5 can be found at the dedicated product page, including references, app notes and more.
The open access article can be found at:

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