Improved mesoscopic imaging - proven in uncleared tumor spheroids

Scientists at the Foundation for Research and Technology – Hellas (FORTH) in Heraklion, Greece have developed a significantly improved methodology for the optical imaging of optically opaque 3D cell cultures with enhanced resolution.

The so-called Phase-Retrieved Tomography (PRT) utilises a Light Sheet / Selective Plane Illumination Microscope (SPIM), Optical Projection Tomography and algorithms to solve challenges around autocorrelation.  PRT’s ability to permit 3D image reconstruction was tested on unfixed, uncleared multicellular tumor spheroids of 200 µm diameter for early stage necrosis using a fluorescent viability dye as a representative and practically relevant proof of concept for the technology.

In the model system individual tumor spheroids were grown in hanging-drop cultures, and further incubated with the addition of the far-red viability dye DRAQ7 for 24 hours to mark the presence of membrane-compromised dead cells.

After a wash procedure a single spheroid was then transferred into CyGEL Sustain, a thermo-reversible hydrogel mountant compatible with living cells and tissues, and this combination placed in a FEP tube, which was sealed and placed in a 37 °C waterbath for the duration of the imaging procedure.

Reference: 

Ancora, D., Di Battista, D., Giasafaki, G., Psycharakis, S. E., Liapis, E., Ripoll, J., & Zacharakis, G. (2017). Phase-Retrieved Tomography enables Mesoscopic imaging of Opaque Tumor Spheroids. 

Scientific Reports, 7(1),11854 (open acess)

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New Guidelines for Cytometry and Sorting in Immunology

Download this open access 200-page article written by 231 co-authors, experts in their respective fields, aimed at all who use flow cytometry in their immunological research to be better equipped to achieve their experimental goals and be publication-ready.

The European Journal of Immunology (EJI) already insists upon the inclusion of gating strategies in submitted manuscripts and, therefore, it is highly appropriate that this 2-year long effort was headed by expert immunologists with editorial responsibilities at EJI and a strong commitment to the advancement of cytometric technologies.

These comprehensive guidelines include sections on equipment set-up, data acquisition & handling, barcoding, CyTOF, combinatorial & imaging cytometry, complete immune cell cytometric phenotypes, and much more.

Our far-red viability probe DRAQ7™ gets a prominent mention in the section on “Cell Death”, with particular reference to its ability to open up an additional “virtual” channel for the exclusion of dead cells in both analysis and sorting.

The issue can be found here:

http://onlinelibrary.wiley.com/doi/10.1002/eji.v47.10/issuetoc 

and direct links to the guidelines: 

PDF http://onlinelibrary.wiley.com/doi/10.1002/eji.201646632/epdf 

HTML http://onlinelibrary.wiley.com/doi/10.1002/eji.201646632/full

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Macrophage cell therapy for cirrhosis

Scientists at the MRC Centre for Regenerative Medicine and the Scottish National Blood Transfusion Centre in Edinburgh have worked together on GMP-compliant manufacture for a novel approach using autologous phagocytic macrophages as a therapeutic approach to mitigate and reverse liver damage in cirrhosis.

As part of the extensive evaluation, in one part they characterised the resulting cells for release criteria and this included DRAQ7(TM) to establish the cell viability alongside the phenotypic marker panel to define the committed phagocytic macrophages: CD45, CD206, CD14 & 25F9.  In their protocol, cells were incubated with FcR block before addition of antibody cocktails, subsequent washing with PEA buffer and addition of DRAQ7 at 3 µM final concentration.

Flow cytometry was performed on both the FACSCanto II and the MACQuant 10. The antibody-chromophore (BV421, FITC, PE, APC) / DRAQ7 panel was compatible with both analysis platforms.  The emission spectrum of DRAQ7 is such that spillover is limited and only in the APC channel.*

* This spillover and requirement for compensation can be completely abrogated by application of the virtual channel method that DRAQ7 uniquely permits. For example, with the chromophores used above, DRAQ7-positive dual-excited events can be displayed in a bivariate plot such as FL3 (PerCP-Cy5.5) vs FL6 (APC-Cy7) or FL4 (PE-Cy7) vs FL5 (APC) in the case of the FACSCanto II. A full explanation of this can be found at www.biostatus.com/DRAQ7 and has been the subject of a poster at ESCCA's annual conference in September 2017.

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Reference:

Fraser, Alasdair R., et al.
"Development, functional characterization and validation of methodology for GMP-compliant manufacture of phagocytic macrophages: A novel cellular therapeutic for liver cirrhosis."
Cytotherapy 19.9 (2017): 1113-1124.