Rmax: A systematic approach to evaluate instrument sort performance using center stream catch.

Sorting performance can be evaluated with regard to Purity, Yield and/or Recovery of the sorted fraction. Purity is a check on the quality of the sample and the sort decisions made by the instrument. Recovery and Yield definitions vary with some authors regarding both as how efficient the instrument is at sorting the target particles from the original sample, others distinguishing Recovery from Yield, where the former is used to describe the accuracy of the instrument's sort count. Yield and Recovery are often neglected, mostly due to difficulties in their measurement. Purity of the sort product is often cited alone but is not sufficient to evaluate sorting performance. All of these three performance metrics require re-sampling of the sorted fraction. But, unlike Purity, calculating Yield and/or Recovery calls for the absolute counting of particles in the sorted fraction, which may not be feasible, particularly when dealing with rare populations and precious samples. In addition, the counting process itself involves large errors. Here we describe a new metric for evaluating instrument sort Recovery, defined as the number of particles sorted relative to the number of original particles to be sorted. This calculation requires only measuring the ratios of target and non-target populations in the original pre-sort sample and in the waste stream or center stream catch (CSC), avoiding re-sampling the sorted fraction and absolute counting. We called this new metric Rmax, since it corresponds to the maximum expected Recovery for a particular set of instrument parameters. Rmax is ideal to evaluate and troubleshoot the optimum drop-charge delay of the sorter, or any instrument related failures that will affect sort performance. It can be used as a daily quality control check but can be particularly useful to assess instrument performance before single-cell sorting experiments. Because we do not perturb the sort fraction we can calculate Rmax during the sort process, being especially valuable to check instrument performance during rare population sorts.Andrew Riddell is based at the Wellcome Trust-MRC Stem Cell Institute, Centre for Stem Cell Research University of CambridgeThis is the final published version. It first appeared at http://www.sciencedirect.com/science/article/pii/S104620231500090

Double-walled poly-(D,l-lactide-co-glycolide) (plga) and poly(l-lactide) (plla) nanoparticles for the sustained release of doxorubicin

Funding Information: This research was funded by Funda??o para a Ci?ncia e a Tecnologia, Portugal, [grant numbers PTDC/QUE/EPR/119631/2010, SFRH/BD/48773/2008] and by the Associate Laboratory for Green Chemistry-LAQV which is financed by national funds from FCT/MCTES [UIDB/50006/2020 and UIDP/50006/2020] and co-financed by the ERDF [PT2020 Partnership Agreement POCI-01-0145-FEDER-007265].Double-walled nanoparticles (DWNPs), containing doxorubicin as a model drug, were produced using poly-(D,L-lactide-co-glycolide) (PLGA) and poly(L-lactide) (PLLA) by the solvent evaporation technique. Double-walled microparticles containing doxorubicin were also produced to make possible the examination of the inner morphology and drug distribution using optical and fluorescence microscopy. The produced microparticles present a double-walled structure with doxorubicin solubilized in the PLGA-rich phase. The DWNPs produced present very low initial burst values and a sustained DOX release for at least 90 days with release rates decreasing with the increase in the PLLA amount. Zero-order release kinetics were obtained after day 15. The results support that the PLLA layer acts as a rate control barrier and that the diffusion of doxorubicin from the drug-loaded inner PLGA core can be retarded by an increase in the thickness of the unloaded outer layer. The unloaded double-walled nanoparticles produced were used in in vitro tests with CHO cells and demonstrate that they are nontoxic, while the double-walled nanoparticles loaded with doxorubicin caused a great cellular viability and decreased when tested in vitro.publishersversionpublishe

Targeting the hemangioblast with a novel cell type-specific enhancer

<p>Abstract</p> <p>Background</p> <p>Hemangioblasts are known as the common precursors for primitive hematopoietic and endothelial lineages. Their existence has been supported mainly by the observation that both cell types develop in close proximity and by in vitro differentiation and genetic studies. However, more compelling evidence will arise from tracking their cell fates using a lineage-specific marker.</p> <p>Results</p> <p>We report the identification of a hemangioblast-specific enhancer (Hb) located in the <it>cis</it>-regulatory region of chick <it>Cerberus </it>gene (<it>cCer</it>) that is able to direct the expression of enhanced green fluorescent protein (eGFP) to the precursors of yolk sac blood and endothelial cells in electroporated chick embryos. Moreover, we present the Hb-eGFP reporter as a powerful live imaging tool for visualizing hemangioblast cell fate and blood island morphogenesis.</p> <p>Conclusions</p> <p>We hereby introduce the Hb enhancer as a valuable resource for genetically targeting the hemangioblast population as well as for studying the dynamics of vascular and blood cell development.</p

A Novel Quantitative Fluorescent Reporter Assay for RAG Targets and RAG Activity

Recombination-Activating Genes (RAG) 1 and 2 form the site specific recombinase that mediates V(D)J recombination, a process of DNA editing required for lymphocyte development and responsible for their diverse repertoire of antigen receptors. Mistargeted RAG activity associates with genome alteration and is responsible for various lymphoid tumors. Moreover several non-lymphoid tumors express RAG ectopically. A practical and powerful tool to perform quantitative assessment of RAG activity and to score putative RAG-Recognition signal sequences (RSS) is required in the fields of immunology, oncology, gene therapy, and development. Here we report the detailed characterization of a novel fluorescence-based reporter of RAG activity, named GFPi, a tool that allows measuring recombination efficiency (RE) by simple flow cytometry analysis. GFPi can be produced both as a plasmid for transient transfection experiments in cell lines or as a retrovirus for stable integration in the genome, thus supporting ex vivo and in vivo studies. The GFPi assay faithfully quantified endogenous and ectopic RAG activity as tested in genetically modified fibroblasts, tumor derived cell lines, developing pre-B cells, and hematopoietic cells. The GFPi assay also successfully ranked the RE of various RSS pairs, including bona fide RSS associated with V(D)J segments, artificial consensus sequences modified or not at specific nucleotides known to affect their efficiencies, or cryptic RSS involved in RAG-dependent activation of oncogenes. Our work validates the GFPi reporter as a practical quantitative tool for the study of RAG activity and RSS efficiencies. It should turn useful for the study of RAG-mediated V(D)J and aberrant rearrangements, lineage commitment, and vertebrate evolution.Portuguese League Against Cancer, Terry Fox Foundation, FCT fellowships

Ploidy mosaicism and allele-specific gene expression differences in the allopolyploid Squalius alburnoides

<p>Abstract</p> <p>Background</p> <p><it>Squalius alburnoides </it>is an Iberian cyprinid fish resulting from an interspecific hybridisation between <it>Squalius pyrenaicus </it>females (P genome) and males of an unknown <it>Anaecypris hispanica-</it>like species (A genome). <it>S. alburnoides </it>is an allopolyploid hybridogenetic complex, which makes it a likely candidate for ploidy mosaicism occurrence, and is also an interesting model to address questions about gene expression regulation and genomic interactions. Indeed, it was previously suggested that in <it>S. alburnoides </it>triploids (PAA composition) silencing of one of the three alleles (mainly of the P allele) occurs. However, not a whole haplome is inactivated but a more or less random inactivation of alleles varying between individuals and even between organs of the same fish was seen.</p> <p>In this work we intended to correlate expression differences between individuals and/or between organs to the occurrence of mosaicism, evaluating if mosaics could explain previous observations and its impact on the assessment of gene expression patterns.</p> <p>Results</p> <p>To achieve our goal, we developed flow cytometry and cell sorting protocols for this system generating more homogenous cellular and transcriptional samples. With this set-up we detected 10% ploidy mosaicism within the <it>S. alburnoides </it>complex, and determined the allelic expression profiles of ubiquitously expressed genes (<it>rpl8</it>; <it>gapdh </it>and <it>β-actin</it>) in cells from liver and kidney of mosaic and non-mosaic individuals coming from different rivers over a wide geographic range.</p> <p>Conclusions</p> <p>Ploidy mosaicism occurs sporadically within the <it>S. alburnoides </it>complex, but in a frequency significantly higher than reported for other organisms. Moreover, we could exclude the influence of this phenomenon on the detection of variable allelic expression profiles of ubiquitously expressed genes (<it>rpl8</it>; <it>gapdh </it>and <it>β-actin</it>) in cells from liver and kidney of triploid individuals. Finally, we determined that the expression patterns previously detected only in a narrow geographic range is not a local restricted phenomenon but is pervasive in rivers where <it>S. pyrenaicus </it>is sympatric with <it>S. alburnoides</it>.</p> <p>We discuss mechanisms that could lead to the formation of mosaic <it>S. alburnoides </it>and hypothesise about a relaxation of the mechanisms that impose a tight control over mitosis and ploidy control in mixoploids.</p

FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei

Background: The male germline in flowering plants differentiates by asymmetric division of haploid uninucleated microspores, giving rise to a vegetative cell enclosing a smaller generative cell, which eventually undergoes a second mitosis to originate two sperm cells. The vegetative cell and the sperm cells activate distinct genetic and epigenetic mechanisms to control pollen tube growth and germ cell specification, respectively. Therefore, a comprehensive characterization of these processes relies on efficient methods to isolate each of the different cell types throughout male gametogenesis. Results: We developed stable transgenic Arabidopsis lines and reliable purification tools based on Fluorescence-Activated Cell Sorting (FACS) in order to isolate highly pure and viable fractions of each cell/nuclei type before and after pollen mitosis. In the case of mature pollen, this was accomplished by expressing GFP and RFP in the sperm and vegetative nuclei, respectively, resulting in 99% pure sorted populations. Microspores were also purified by FACS taking advantage of their characteristic small size and autofluorescent properties, and were confirmed to be 98% pure. Conclusions: We provide simple and efficient FACS-based purification protocols for Arabidopsis microspores, vegetative nuclei and sperm cells. This paves the way for subsequent molecular analysis such as transcriptomics, DNA methylation analysis and chromatin immunoprecipitation, in the developmental context of microgametogenesis in Arabidopsis.NSERC graduate student fellowship; Fred C. Gloeckner Foundation grant

Characterization of Coelomic Fluid Cell Types in the Starfish Marthasterias glacialis Using a Flow Cytometry/Imaging Combined Approach

Funding: To the “Maristem COST Action” (CA16203), supported by COST (European Cooperation in Science and Technology), for funding PM STSM visits (February 2019 and November 2020) to the AVC Laboratory.Coelomocytes is the generic name for a collection of cellular morphotypes, present in many coelomate animals, and highly variable among echinoderm classes. The roles attributed to the major types of these free circulating cells present in the coelomic fluid of echinoderms include immune response, phagocytic digestion and clotting. Our main aim in this study was to characterize coelomocytes found in the coelomic fluid of Marthasterias glacialis (class Asteroidea) by using a combination of flow cytometry (FC), imaging flow cytometry (IFC) and fluorescence plus transmission electron microscopy (TEM). Two coelomocyte populations (P1 and P2) identified through flow cytometry were subsequently studied in terms of abundance, morphology, ultrastructure, cell viability and cell cycle profiles. Ultrastructurally, P2 diploid cells were present as two main morphotypes, similar to phagocytes and vertebrate thrombocytes, whereas the smaller P1 cellular population was characterized by low mitotic activity, a relatively undifferentiated cytotype and a high nucleus/cytoplasm ratio. In the present study we could not rule out possible similarities between haploid P1 cells and stem-cell types in other animals. Additionally, we report the presence of two other morphotypes in P2 that could only be detected by fluorescence microscopy, as well as a morphotype revealed via combined microscopy/FC. This integrative experimental workflow combined cells physical separation with different microscopic image capture technologies, enabling us to better tackle the characterization of the heterogeneous composition of coelomocytes populations.publishersversionpublishe

Single-shot 3D coherent diffractive imaging of core-shell nanoparticles with elemental specificity.

We report 3D coherent diffractive imaging (CDI) of Au/Pd core-shell nanoparticles with 6.1 nm spatial resolution with elemental specificity. We measured single-shot diffraction patterns of the nanoparticles using intense x-ray free electron laser pulses. By exploiting the curvature of the Ewald sphere and the symmetry of the nanoparticle, we reconstructed the 3D electron density of 34 core-shell structures from these diffraction patterns. To extract 3D structural information beyond the diffraction signal, we implemented a super-resolution technique by taking advantage of CDI's quantitative reconstruction capabilities. We used high-resolution model fitting to determine the Au core size and the Pd shell thickness to be 65.0 ± 1.0 nm and 4.0 ± 0.5 nm, respectively. We also identified the 3D elemental distribution inside the nanoparticles with an accuracy of 3%. To further examine the model fitting procedure, we simulated noisy diffraction patterns from a Au/Pd core-shell model and a solid Au model and confirmed the validity of the method. We anticipate this super-resolution CDI method can be generally used for quantitative 3D imaging of symmetrical nanostructures with elemental specificity