DRAQ5 uncovers life-cycle of Microsporidia pathogen

Scientists at Rutgers University and the National Institutes of Health have recently developed novel fluorescence-based methods for the tracking of the microsporidial pathogenic parasite Anncaliia algerae; its development in the human host cellular niche

A. algerae is an opportunistic parasitic pathogen that is a risk to immune-compromised individuals and was first identified in mosquitoes, that is however poorly understood due to limited methodologies.

The far-red cell permeant DNA probe DRAQ5 was used as the key fluorescent reagent to label live parasites prior to infection of, and time-lapse imaging inside, GFP-transformed HeLa cells observed for 8 days.  The DRAQ5 stained A. algerae inoculum successfully infected the host cells and produced numerous progeny via unaffected mitosis.

DRAQ5 was also applied for conventional fixed cell end-point imaging of infected host cells.

These studies should hopefully increase understanding of the biology and life-cycle of pathogenic microsporidia sp.

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Read the J. Euk. Microbiol. paper here: http://tinyurl.com/zw4xvd9

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.

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The open access article can be found at:  http://tinyurl.com/z48z26j

Context-Dependent Cell Population Dynamics

NEW DRAQ7 ARTICLE: Investigation of complex cell population dynamics

Garvey et al. (Scientific Reports 6:29752; Published 25th July, 2016; Open access) from USC, UMN & Stanford describe a high-content imaging approach to monitoring heterogeneous cell population dynamics in the context of time and different possibly co-occurring selective pressures such as therapy, hypoxia, microenvironment and so on. For example, they were able to differentiate between cytostatic and cytotoxic effects of anti-cancer compounds on cell mixtures with different sensitivities that would have been masked by bulk measurements of MTS, ATP or total cell numbers. They demonstrate their approach in both 2-D and 3-D culture systems and suggest it would be amenable to any lab with a live-cell imaging platform (not only HCS equipment), utilising open-source software or proprietary alternatives. What they propose is a synthesis of mostly pre-existing techniques reminding us of the complexity of the systems we are trying to mimic in in vitro cancer cell biology!

In some examples they combine Hoechst 33342 (all nucleated cells) and DRAQ7 (dead/leaky cells) to monitor cell population death, stasis and expansion for 2-D culture and 3-D spheroids composed of two isogenically-identical cell types differing only in their sensitivity to chemotherapy and marked accordingly by constitutive GFP and RFP expression.

To show the limitations of detachment of adherent cells for analysis by flow cytometry they used the same reagent combination.

The far-red viability probe DRAQ7 demonstrates its cross-platform capabilities and amenability to HCS methodologies.  Though not exploited here, DRAQ7 can also be applied in the culture medium to enable monitoring in time-lapse mode. A white paper on this is available here:
http://tinyurl.com/z7vp5sy

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Reference:
Garvey, C.M., Spiller, E., Lindsay, D., Chiang, C.T., Choi, N.C., Agus, D.B., Mallick, P., Foo, J. and Mumenthaler, S.M., 2016. A high-content image-based method for quantitatively studying context-dependent cell population dynamics. Scientific Reports, 6, p.29752.