Human Embryonic Stem Cells - an ultrastructural analysis of nuclei

Scientists from U Mass Med School have undertaken a detailed study of the nuclei of human embryonic stem cells (hES) to better understand the possible implications of the different features and how these might impact on the likely pluripotence of these cells.

The far-red DNA binding probe DRAQ5 was deployed by the authors to observe the expression and presence of nuclear pores consistent with the appropriate phenotype, but not a residue of the source cells after many cell divisions.

To quote the authors: "The architectural organization of human ES cell nuclei has important implications for cell structure – gene expression relationships and for the maintenance of pluripotency."

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iPSC-derived retinal pigment epithelia successfully transplanted

Scientists at RIKEN, Kobe have successfully transplanted MHC-matched iPSC-derived retinal pigment epithelial cells (RPEs) without rejection.

The far-red cell permeant DNA probe DRAQ5 was deployed in an assay to demonstrate the phagocytotic function of the cells to take up FITC-tagged photoreceptor rod outer segment (ROS).  From the DRAQ5 labelling the authors inferred that the cells under analysis were live/intact.

DRAQ5 rapidly and efficiently labels intact nucleated cells with stoichiometry.  DRAQ5 is spectrally separated from FITC.  It enables simplified analysis of complex samples that may contain damaged cells and debris while limiting the requirement for sample preparation.

The research in this work points to the possibility of successful transplantation of cells from MHC homozygous donors might be used to treat retinal diseases in histocompatible patients.

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Keeping score on centromeres with DRAQ5

Scientists in the lab of Dr. Ruth Wilkins at Health Canada have further developed their imaging flow cytometry methodologies for radiation biodosimetry, to detect the presence of aberrant dicentric chromosomes as a further tool to improve the ability to triage exposed individuals in the event of a large-scale radiological or nuclear emergency, such as the Fukushima disaster. 

This work builds on their earlier publications using the far-red cell permeant DNA probe DRAQ5 (BioStatus Limited) to detect micronuclei as a facile signature for radiation damage in peripheral blood leukocytes on the Amnis Imagestream imaging flow cytometer (EMD-Millipore).

Briefly, in this most recent work, they isolate leukocytes from fresh blood, stimulate the cells with PHA and interfere with proper chromosome separation with colcemid to promote formation of dicentric chromosomes. The chromosomes are released into suspension and labeled with a centromere-specific fluorescent PNA probe and DRAQ5 for DNA content. The labeled chromosomes are analysed by imaging flow cytometry to measure the centromere count per chromosome.

Reference: Beaton-Green LA, Rodrigues MA, Lachapelle S, Wilkins RC. Foundations of identifying individual chromosomes by imaging flow cytometry with applications in radiation biodosimetry. Methods. 2016 Aug 12.

Quick link to the article: http://tinyurl.com/zkcbwom

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Anti-tumour vaccine research (CDC assay) utilises DRAQ7

French scientists have shown use of a tetanus toxoid (TT) peptide sequence with a tumour multi-antigenic glycopeptide (MAG) where the TT sequence acts on T cells to further boost the anti-tumour response.

The work included a complement mediated cytotoxicity (CDC) assay.  The CDC assay was run in real-time (2h) using the far-red viability probe DRAQ7 (at 2 uM) to monitor cell death on the Essen Biosciences Incucyte Zoom.

This work was a collaboration between scientists affiliated with Inst. Pasteur, Inst. Curie, CNRS, INSERM and GSK Vaccines.

Ref:
Laubreton, D., Bay, S., Sedlik, C. et al. Cancer Immunol Immunother (2016) 65: 315. doi:10.1007/s00262-016-1802-0

Quick link to article page

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Cells in Gels: A Disease-Like Morphology for Chondrocytes

In their new article Karim & Hall demonstrate a direct link between gel stiffness and the morphology of chondrocytes. 

In soft gels cellular morphology (density, volume) and clustering is more akin to that seen in osteoarthritis (OA). Increased concentrations of fetal calf serum (FCS) further influenced this effect.

The authors surmise that the softer gels might offer higher permeance for soluble factors in the FCS that lead to the observed changes.

These findings may be important in understanding the impact of ECM on OA and related cartilage damage as well as for tissue engineering.

The far-red DNA probe DRAQ5 was used to aid the cellular volume measurements alongside propidium iodide and calculated in Volocity(TM) software.

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Article:

Karim, Asima, and Andrew C. Hall. "Chondrocyte Morphology in Stiff and Soft Agarose Gels and the Influence of Fetal Calf Serum." Journal of Cellular Physiology (2016). 

Read it here:
http://tinyurl.com/gmcp25p

Babraham Inst. Flow Cytometry Courses - Autumn 2016/Spring 2017 dates

The Babraham Institute has announced the dates for Autumn 2016 and Spring 2017 of their modular flow cytometry training courses.

These are organised by Rachael Walker, Babraham Institute & Derek Davies, The Francis Crick Institute.  They say places are limited on the courses so sign up soon to avoid disappointment!

Module 1 : Fundamentals of Flow Cytometry
22nd September 2016
20th October 2016
7th February 2017
7th April 2017

Module 2 : Advanced Flow Cytometry
3rd & 4th November 2016
8th & 9th March 2017

For more details and to register please go to: 
http://www.babraham.ac.uk/science-services/flow-cytometry/flow-training