A novel flow cytometric method to assess inflammasome formation.

Inflammasomes are large protein complexes induced by a wide range of microbial, stress, and environmental stimuli that function to induce cell death and inflammatory cytokine processing. Formation of an inflammasome involves dramatic relocalization of the inflammasome adapter protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) into a single speck. We have developed a flow cytometric assay for inflammasome formation, time of flight inflammasome evaluation, which detects the change in ASC distribution within the cell. The transit of ASC into the speck is detected by a decreased width or increased height of the pulse of emitted fluorescence. This assay can be used to quantify native inflammasome formation in subsets of mixed cell populations ex vivo. It can also provide a rapid and sensitive technique for investigating molecular interactions in inflammasome formation, by comparison of wild-type and mutant proteins in inflammasome reconstitution experiments.

Purification of neural precursor cells reveals the presence of distinct, stimulus-specific subpopulations of quiescent precursors in the adult mouse hippocampus.

The activity of neural precursor cells in the adult hippocampus is regulated by various stimuli; however, whether these stimuli regulate the same or different precursor populations remains unknown. Here, we developed a novel cell-sorting protocol that allows the purification to homogeneity of neurosphere-forming neural precursors from the adult mouse hippocampus and examined the responsiveness of individual precursors to various stimuli using a clonal assay. We show that within the Hes5-GFP(+)/Nestin-GFP(+)/EGFR(+) cell population, which comprises the majority of neurosphere-forming precursors, there are two distinct subpopulations of quiescent precursor cells, one directly activated by high-KCl depolarization, and the other activated by norepinephrine (NE). We then demonstrate that these two populations are differentially distributed along the septotemporal axis of the hippocampus, and show that the NE-responsive precursors are selectively regulated by GABA, whereas the KCl-responsive precursors are selectively modulated by corticosterone. Finally, based on RNAseq analysis by deep sequencing, we show that the progeny generated by activating NE-responsive versus KCl-responsive quiescent precursors are molecularly different. These results demonstrate that the adult hippocampus contains phenotypically similar but stimulus-specific populations of quiescent precursors, which may give rise to neural progeny with different functional capacity.

Development of a novel cell sorting method that samples population diversity in flow cytometry.

Flow cytometry based electrostatic cell sorting is an important tool in the separation of cell populations. Existing instruments can sort single cells into multi-well collection plates, and keep track of cell of origin and sorted well location. However currently single sorted cell results reflect the population distribution and fail to capture the population diversity. Software was designed that implements a novel sorting approach, "Slice and Dice Sorting," that links a graphical representation of a multi-well plate to logic that ensures that single cells are sampled and sorted from all areas defined by the sort region/s. Therefore the diversity of the total population is captured, and the more frequently occurring or rarer cell types are all sampled. The sorting approach was tested computationally, and using functional cell based assays. Computationally we demonstrate that conventional single cell sorting can sample as little as 50% of the population diversity dependant on the population distribution, and that Slice and Dice sorting samples much more of the variety present within a cell population. We then show by sorting single cells into wells using the Slice and Dice sorting method that there are cells sorted using this method that would be either rarely sorted, or not sorted at all using conventional single cell sorting approaches. The present study demonstrates a novel single cell sorting method that samples much more of the population diversity than current methods. It has implications in clonal selection, stem cell sorting, single cell sequencing and any areas where population heterogeneity is of importance.

Absolute counting of neutrophils in whole blood using flow cytometry.

Absolute neutrophil count (ANC) is used clinically to monitor physiological dysfunctions such as myelosuppression or infection. In the research laboratory, ANC is a valuable measure to monitor the evolution of a wide range of disease states in disease models. Flow cytometry (FCM) is a fast, widely used approach to confidently identify thousands of cells within minutes. FCM can be optimised for absolute counting using spiked-in beads or by measuring the sample volume analysed. Here we combine the 1A8 antibody, specific for the mouse granulocyte protein Ly6G, with flow cytometric counting in straightforward FCM assays for mouse ANC, easily implementable in the research laboratory. Volumetric and Trucount™ bead assays were optimized for mouse neutrophils, and ANC values obtained with these protocols were compared to ANC measured by a dual-platform assay using the Orphee Mythic 18 veterinary haematology analyser. The single platform assays were more precise with decreased intra-assay variability compared with ANC obtained using the dual protocol. Defining ANC based on Ly6G expression produces a 15% higher estimate than the dual protocol. Allowing for this difference in ANC definition, the flow cytometry counting assays using Ly6G can be used reliably in the research laboratory to quantify mouse ANC from a small volume of blood. We demonstrate the utility of the volumetric protocol in a time-course study of chemotherapy induced neutropenia using four drug regimens.

Effect of a hemiuroid trematode on the hemocyte immune parameters of the cockle Anadara trapezia.

When a trematode parasite penetrates a potential molluscan host, it has to circumvent the host's internal defense system. In molluscs, the primary effector cells of this system are the hemocytes which orchestrate many of the cellular and humoral immune functions. Survival of the parasite can occur only in the absence of a successful immune response, and continued development only if the host is physiologically suitable. This study investigated hemocytic response against asexual stages of a hemiuroid trematode by its host, the marine bivalve Anadara trapezia. Hemocyte characteristic (type, morphology) and function (mortality, phagocytosis and oxidative activity) were analyzed by flow cytometry in parasitized and non-parasitized cockles. A. trapezia possesses two types of hemocytes: amebocytes and erythrocytes. Analysis of histological section showed that there was no host hemocytic response around hemiuroid sporocysts. The infection induced a significant increase of the total circulating hemocytes with a higher proportion of erythrocytes relative to amebocytes, coupled with a lower phagocytosis rate and a statistically non-significant decrease of the intracellular oxidative activity. No significant differences were observed in hemocyte size and complexity, mortality, or phagocytic capacity. Our results indicate that in A. trapezia, hemiuroids modulate the immune response by increasing the number of circulating hemocytes and decreasing phagocytosis.

Evidence for label-retaining tumour-initiating cells in human glioblastoma.

Individual tumour cells display diverse functional behaviours in terms of proliferation rate, cell-cell interactions, metastatic potential and sensitivity to therapy. Moreover, sequencing studies have demonstrated surprising levels of genetic diversity between individual patient tumours of the same type. Tumour heterogeneity presents a significant therapeutic challenge as diverse cell types within a tumour can respond differently to therapies, and inter-patient heterogeneity may prevent the development of general treatments for cancer. One strategy that may help overcome tumour heterogeneity is the identification of tumour sub-populations that drive specific disease pathologies for the development of therapies targeting these clinically relevant sub-populations. Here, we have identified a dye-retaining brain tumour population that displays all the hallmarks of a tumour-initiating sub-population. Using a limiting dilution transplantation assay in immunocompromised mice, label-retaining brain tumour cells display elevated tumour-initiation properties relative to the bulk population. Importantly, tumours generated from these label-retaining cells exhibit all the pathological features of the primary disease. Together, these findings confirm dye-retaining brain tumour cells exhibit tumour-initiation ability and are therefore viable targets for the development of therapeutics targeting this sub-population.

Cellular plasticity of inflammatory myeloid cells in the peritoneal foreign body response.

Implantation of sterile foreign objects in the peritoneal cavity of an animal initiates an inflammatory response and results in encapsulation of the objects by bone marrow-derived cells. Over time, a multilayered tissue capsule develops with abundant myofibroblasts embedded in extracellular matrix. The present study used the transgenic MacGreen mouse to characterize the time-dependent accumulation of monocyte subsets and neutrophilic granulocytes in the inflammatory infiltrate and within the tissue capsule by their differential expression of the csf1r-EGFP transgene, F4/80, and Ly6C. As the tissue capsule developed, enhanced green fluorescent protein-positive cells changed from rounded to spindle-shaped morphology and began to co-express the myofibroblast marker alpha-smooth muscle actin. Expression increased with time: at day 14, 11.13 +/- 0.67% of tissue capsule cells co-expressed these markers, compared with 50.77 +/- 12.85% of cells at day 28. The importance of monocyte/macrophages in tissue capsule development was confirmed by clodronate-encapsulated liposome removal, which resulted in almost complete abrogation of capsule development. These results confirm the importance of monocyte/macrophages in the tissue response to sterile foreign objects implanted in the peritoneal cavity. In addition, the in vivo plasticity of peritoneal macrophages and their ability to transdifferentiate from a myeloid to mesenchymal phenotype is demonstrated.

A method of quantifying cell sorting yield in "real time".

Cell sorting flow cytometers sort cells by applying electrical charges to a stream that forms liquid droplets containing the cells at a set time after sample interrogation. The correct time to apply these charges is determined based on calibration beads and automated technology. The central tenet of this method is that beads accurately indicate the yield of cells sorted using the same instrument parameters. HEK293T cells were incubated with Accudrop™ calibration beads. Cell incorporation of beads by phagocytosis was confirmed by imaging cytometry. Cells containing beads were analyzed and sorted on unmodified commercially available cell sorters that have automated technology for setting drop delay times. Based on cell sorter drop delay times optimized using beads, sorting experiments demonstrate that yield can be assessed in real time using bead loaded cells and existing technology. Here, data presented show that cells have lower sorting yields than indicated using beads. Further, this data show clear trends related to nozzle tip diameter and sorting yield. The present study demonstrates a method to quantify yield "on the fly" during cell sorting, that is separated from drop charge counts and removes the variables associated with yield assessment from collection tubes. These data have implications for the expected recovery of cells from sorting experiments.

p75 neurotrophin receptor regulates basal and fluoxetine-stimulated hippocampal neurogenesis.

It is widely acknowledged that neurogenesis occurs in the adult hippocampus under normal conditions and that the rate can be regulated by environmental factors, including antidepressant drugs, with concomitant effects on behaviour. Using a quick and sensitive flow cytometry method that can assess changes in the number of bromodeoxyuridine (BrdU)-positive cells in hippocampus, in combination with traditional histological cell counts in the dentate gyrus, we report that mice lacking the p75 neurotrophin receptor gene (p75(NTR-/-)) have significantly reduced hippocampal neurogenesis. Chronic treatment with the antidepressant fluoxetine stimulated hippocampal cell proliferation in p75(NTR-/-) animals, but it did not result in an increase above basal levels of the number of newly born neurons in the dentate gyrus. These results indicate that p75(NTR) acts as a regulator of fluoxetine-stimulated as well as basal adult hippocampal neurogenesis.

Multiplexed labeling of viable cells for high-throughput analysis of glycine receptor function using flow cytometry.

Flow cytometry is an important drug discovery tool because it permits high-content multiparameter analysis of individual cells. A new method dramatically enhanced screening throughput by multiplexing many discrete fixed cell populations; however, this method is not suited to assays requiring functional cellular responses. HEK293 cells were transfected with unique mutant glycine receptors. Mutant receptor expression was confirmed by coexpression of yellow fluorescent protein (YFP). Commercially available cell-permeant dyes were used to label each glycine receptor expressing mutant with a unique optical code. All encoded cell lines were combined in a single tube and analyzed on a flow cytometer simultaneously before and after the addition of glycine receptor agonist. We decoded multiplexed cells that expressed functionally distinct glycine receptor chloride channels and analyzed responses to glycine in terms of chloride-sensitive YFP expression. Here, data provided by flow cytometry can be used to discriminate between functional and nonfunctional mutations in the glycine receptor, a process accelerated by the use of multiplexing. Further, this data correlates to data generated using a microscopy-based technique. The present study demonstrates multiplexed labeling of live cells, to enable cell populations to be subject to further cell culture and experimentation, and compares the results with those obtained using live cell microscopy.

Identification of CD24 as a marker of Patched1 deleted medulloblastoma-initiating neural progenitor cells.

High morbidity and mortality are common traits of malignant tumours and identification of the cells responsible is a focus of on-going research. Many studies are now reporting the use of antibodies specific to Clusters of Differentiation (CD) cell surface antigens to identify tumour-initiating cell (TIC) populations in neural tumours. Medulloblastoma is one of the most common malignant brain tumours in children and despite a considerable amount of research investigating this tumour, the identity of the TICs, and the means by which such cells can be targeted remain largely unknown. Current prognostication and stratification of medulloblastoma using clinical factors, histology and genetic profiling have classified this tumour into four main subgroups: WNT, Sonic hedgehog (SHH), Group 3 and Group 4. Of these subgroups, SHH remains one of the most studied tumour groups due to the ability to model medulloblastoma formation through targeted deletion of the Shh pathway inhibitor Patched1 (Ptch1). Here we sought to utilise CD antibody expression to identify and isolate TIC populations in Ptch1 deleted medulloblastoma, and determine if these antibodies can help classify the identity of human medulloblastoma subgroups. Using a fluorescence-activated cell sorted (FACS) CD antibody panel, we identified CD24 as a marker of TICs in Ptch1 deleted medulloblastoma. CD24 expression was not correlated with markers of astrocytes or oligodendrocytes, but co-labelled with markers of neural progenitor cells. In conjunction with CD15, proliferating CD24+/CD15+ granule cell precursors (GCPs) were identified as a TIC population in Ptch1 deleted medulloblastoma. On human medulloblastoma, CD24 was found to be highly expressed on Group 3, Group 4 and SHH subgroups compared with the WNT subgroup, which was predominantly positive for CD15, suggesting CD24 is an important marker of non-WNT medulloblastoma initiating cells and a potential therapeutic target in human medulloblastoma. This study reports the use of CD24 and CD15 to isolate a GCP-like TIC population in Ptch1 deleted medulloblastoma, and suggests CD24 expression as a marker to help stratify human WNT tumours from other medulloblastoma subgroups.

Developmental Vitamin D (DVD) Deficiency Reduces Nurr1 and TH Expression in Post-mitotic Dopamine Neurons in Rat Mesencephalon.

Developmental vitamin D (DVD) deficiency has been proposed as an important risk factor for schizophrenia. Our previous study using Sprague Dawley rats found that DVD deficiency disrupted the ontogeny of mesencephalic dopamine neurons by decreasing the mRNA level of a crucial differentiation factor of dopamine cells, the nuclear receptor related 1 protein (Nurr1). However, it remains unknown whether this reflects a reduction in dopamine cell number or in Nurr1 expression. It is also unclear if any particular subset of developing dopamine neurons in the mesencephalon is selectively affected. In this study, we employed state-of-the-art spinning disk confocal microscopy optimized for the imaging of tissue sections and 3D segmentation to assess post-mitotic dopamine cells on a single-cell basis in the rat mesencephalon at embryonic day 15. Our results showed that DVD deficiency did not alter the number, morphology, or positioning of post-mitotic dopamine cells. However, the ratio of Nurr1+TH+ cells in the substantia nigra pars compacta (SNc) compared with the ventral tegmental area (VTA) was increased in DVD-deficient embryos. In addition, the expression of Nurr1 in immature dopamine cells and mature dopamine neurons in the VTA was decreased in DVD-deficient group. Tyrosine hydroxylase was selectively reduced in SNc of DVD-deficient mesencephalon. We conclude that DVD deficiency induced early alterations in mesencephalic dopamine development may in part explain the abnormal dopamine-related behaviors found in this model. Our findings may have broader implications for how certain environmental risk factors for schizophrenia may shape the ontogeny of dopaminergic systems and by inference increase the risk of schizophrenia.

Functional domain analysis of SOX18 transcription factor using a single-chain variable fragment-based approach.

Antibodies are routinely used to study the activity of transcription factors, using various in vitro and in vivo approaches such as electrophoretic mobility shift assay, enzyme-linked immunosorbent assay, genome-wide method analysis coupled with next generation sequencing, or mass spectrometry. More recently, a new application for antibodies has emerged as crystallisation scaffolds for difficult to crystallise proteins, such as transcription factors. Only in a few rare cases, antibodies have been used to modulate the activity of transcription factors, and there is a real gap in our knowledge on how to efficiently design antibodies to interfere with transcription. The molecular function of transcription factors is underpinned by complex networks of protein-protein interaction and in theory, setting aside intra-cellular delivery challenges, developing antibody-based approaches to modulate transcription factor activity appears a viable option. Here, we demonstrate that antibodies or an antibody single-chain variable region fragments are powerful molecular tools to unravel complex protein-DNA and protein-protein binding mechanisms. In this study, we focus on the molecular mode of action of the transcription factor SOX18, a key modulator of endothelial cell fate during development, as well as an attractive target in certain pathophysiological conditions such as solid cancer metastasis. The engineered antibody we designed inhibits SOX18 transcriptional activity, by interfering specifically with an 8-amino-acid motif in the C-terminal region directly adjacent to α-Helix 3 of SOX18 HMG domain, thereby disrupting protein-protein interaction. This new approach establishes a framework to guide the study of transcription factors interactomes using antibodies as molecular handles.

Targeting membrane proteins for antibody discovery using phage display.

A critical factor in the successful isolation of new antibodies by phage display is the presentation of a correctly folded antigen. While this is relatively simple for soluble proteins which can be purified and immobilized onto a plastic surface, membrane proteins offer significant challenges for antibody discovery. Whole cell panning allows presentation of the membrane protein in its native conformation, but is complicated by a low target antigen density, high background of irrelevant antigens and non-specific binding of phage particles to cell surfaces. The method described here uses transient transfection of alternating host cell lines and stringent washing steps to address each of these limitations. The successful isolation of antibodies from a naive scFv library is described for three membrane bound proteins; human CD83, canine CD117 and bat CD11b.

Flow cytometry of neural cells.

Flow cytometry is an advanced group of techniques for counting and quantifying microscopic particles such as cells, chromosomes, or functionalized beads. These approaches employ sophisticated optical and fluidic components to detect scattered light and fluorescent signals from cells as they sequentially pass an interrogation point. Cytometry plays a crucial role in the diagnosis of immunological disorders and cancers, and is a mainstay technique in basic research settings such as hematology, cell biology, and biomolecular screening. However, in spite of the breadth of applications spanning many fields, flow cytometry in neuroscience has been largely unexploited and has seen only a steady increase in interest until recent years. This is rather surprising as the potential of flow cytometry in neuroscience applications was recognized in the early 1980s as the technology was evolving.

A novel fluorescent reporter CDy1 enriches for neural stem cells derived from the murine brain.

Neurogenesis occurs continuously in two brain regions of adult mammals, underpinned by a pool of resident neural stem cells (NSCs) that can differentiate into all neural cell types. To advance our understanding of NSC function and to develop therapeutic and diagnostic approaches, it is important to accurately identify and enrich for NSCs. There are no definitive markers for the identification and enrichment of NSCs present in the mouse brain. Recently, a fluorescent rosamine dye, CDy1, has been identified as a label for pluripotency in cultured human embryonic and induced pluripotent stem cells. As similar cellular characteristics may enable the uptake and retention of CDy1 by other stem cell populations, we hypothesized that this dye may also enrich for primary NSCs from the mouse brain. Because the subventricular zone (SVZ) and the hippocampus represent brain regions that are highly enriched for NSCs in adult mammals, we sampled cells from these areas to test this hypothesis. These experiments revealed that CDy1 staining indeed allows for enrichment and selection of all neurosphere-forming cells from both the SVZ and the hippocampus. We next examined the effectiveness of CDy1 to select for NSCs derived from the SVZ of aged animals, where the total pool of NSCs present is significantly lower than in young animals. We found that CDy1 effectively labels the NSCs in adult and aged animals as assessed by the neurosphere assay and reflects the numbers of NSCs present in aged animals. CDy1, therefore, appears to be a novel marker for enrichment of NSCs in primary brain tissue preparations.

Glioma surgical aspirate: a viable source of tumor tissue for experimental research.

Brain cancer research has been hampered by a paucity of viable clinical tissue of sufficient quality and quantity for experimental research. This has driven researchers to rely heavily on long term cultured cells which no longer represent the cancers from which they were derived. Resection of brain tumors, particularly at the interface between normal and tumorigenic tissue, can be carried out using an ultrasonic surgical aspirator (CUSA) that deposits liquid (blood and irrigation fluid) and resected tissue into a sterile bottle for disposal. To determine the utility of CUSA-derived glioma tissue for experimental research, we collected 48 CUSA specimen bottles from glioma patients and analyzed both the solid tissue fragments and dissociated tumor cells suspended in the liquid waste fraction. We investigated if these fractions would be useful for analyzing tumor heterogeneity, using IHC and multi-parameter flow cytometry; we also assessed culture generation and orthotopic xenograft potential. Both cell sources proved to be an abundant, highly viable source of live tumor cells for cytometric analysis, animal studies and in-vitro studies. Our findings demonstrate that CUSA tissue represents an abundant viable source to conduct experimental research and to carry out diagnostic analyses by flow cytometry or other molecular diagnostic procedures.

EphA3 maintains tumorigenicity and is a therapeutic target in glioblastoma multiforme.

Significant endeavor has been applied to identify functional therapeutic targets in glioblastoma (GBM) to halt the growth of this aggressive cancer. We show that the receptor tyrosine kinase EphA3 is frequently overexpressed in GBM and, in particular, in the most aggressive mesenchymal subtype. Importantly, EphA3 is highly expressed on the tumor-initiating cell population in glioma and appears critically involved in maintaining tumor cells in a less differentiated state by modulating mitogen-activated protein kinase signaling. EphA3 knockdown or depletion of EphA3-positive tumor cells reduced tumorigenic potential to a degree comparable to treatment with a therapeutic radiolabelled EphA3-specific monoclonal antibody. These results identify EphA3 as a functional, targetable receptor in GBM.

Analysis of mitochondrial function and localisation during human embryonic stem cell differentiation in vitro.

Human embryonic stem cell (hESC) derivatives show promise as viable cell therapy options for multiple disorders in different tissues. Recent advances in stem cell biology have lead to the reliable production and detailed molecular characterisation of a range of cell-types. However, the role of mitochondria during differentiation has yet to be fully elucidated. Mitochondria mediate a cells response to altered energy requirements (e.g. cardiomyocyte contraction) and, as such, the mitochondrial phenotype is likely to change during the dynamic process of hESC differentiation. We demonstrate that manipulating mitochondrial biogenesis alters mesendoderm commitment. To investigate mitochondrial localisation during early lineage specification of hESCs we developed a mitochondrial reporter line, KMEL2, in which sequences encoding the green fluorescent protein (GFP) are targeted to the mitochondria. Differentiation of KMEL2 lines into the three germ layers showed that the mitochondria in these differentiated progeny are GFP positive. Therefore, KMEL2 hESCs facilitate the study of mitochondria in a range of cell types and, importantly, permit real-time analysis of mitochondria via the GFP tag.

Comprehensive transcriptome and immunophenotype analysis of renal and cardiac MSC-like populations supports strong congruence with bone marrow MSC despite maintenance of distinct identities.

Cells resembling bone marrow mesenchymal stem cells (MSC) have been isolated from many organs but their functional relationships have not been thoroughly examined. Here we compared the immunophenotype, gene expression, multipotency and immunosuppressive potential of MSC-like colony-forming cells from adult murine bone marrow (bmMSC), kidney (kCFU-F) and heart (cCFU-F), cultured under uniform conditions. All populations showed classic MSC morphology and in vitro mesodermal multipotency. Of the two solid organ-specific CFU-F, only kCFU-F displayed suppression of T-cell alloreactivity in vitro, albeit to a lesser extent than bmMSC. Quantitative immunophenotyping using 81 phycoerythrin-conjugated CD antibodies demonstrated that all populations contained high percentages of cells expressing diagnostic MSC surface markers (Sca1, CD90.2, CD29, CD44), as well as others noted previously on murine MSC (CD24, CD49e, CD51, CD80, CD81, CD105). Illumina microarray expression profiling and bioinformatic analysis indicated a correlation of gene expression of 0.88-0.92 between pairwise comparisons. All populations expressed approximately 66% of genes in the pluripotency network (Plurinet), presumably reflecting their stem-like character. Furthermore, all populations expressed genes involved in immunomodulation, homing and tissue repair, suggesting these as conserved functions for MSC-like cells in solid organs. Despite this molecular congruence, strong biases in gene and protein expression and pathway activity were seen, suggesting organ-specific functions. Hence, tissue-derived MSC may also retain unique properties potentially rendering them more appropriate as cellular therapeutic agents for their organ of origin.