Residual OXPHOS is required to drive primary and metastatic lung tumours in an orthotopic breast cancer model.

Background: Fast adaptation of glycolytic and mitochondrial energy pathways to changes in the tumour microenvironment is a hallmark of cancer. Purely glycolytic ρ0 tumour cells do not form primary tumours unless they acquire healthy mitochondria from their micro-environment. Here we explored the effects of severely compromised respiration on the metastatic capability of 4T1 mouse breast cancer cells. Methods: 4T1 cell lines with different levels of respiratory capacity were generated; the Seahorse extracellular flux analyser was used to evaluate oxygen consumption rates, fluorescent confocal microscopy to assess the number of SYBR gold-stained mitochondrial DNA nucleoids, and the presence of the ATP5B protein in the cytoplasm and fluorescent in situ nuclear hybridization was used to establish ploidy. MinION nanopore RNA sequence analysis was used to compare mitochondrial DNA transcription between cell lines. Orthotopic injection was used to determine the ability of cells to metastasize to the lungs of female Balb/c mice. Results: OXPHOS-deficient ATP5B-KO3.1 cells did not generate primary tumours. Severely OXPHOS compromised ρ0D5 cells generated both primary tumours and lung metastases. Cells generated from lung metastasis of both OXPHOS-competent and OXPHOS-compromised cells formed primary tumours but no metastases when re-injected into mice. OXPHOS-compromised cells significantly increased their mtDNA content, but this did not result in increased OXPHOS capacity, which was not due to decreased mtDNA transcription. Gene set enrichment analysis suggests that certain cells derived from lung metastases downregulate their epithelial-to-mesenchymal related pathways. Conclusion: In summary, OXPHOS is required for tumorigenesis in this orthotopic mouse breast cancer model but even very low levels of OXPHOS are sufficient to generate both primary tumours and lung metastases.

Modulation of intestinal epithelial permeability by chronic small intestinal helminth infections.

Increased permeability of the intestinal epithelial layer is linked to the pathogenesis and perpetuation of a wide range of intestinal and extra-intestinal diseases. Infecting humans with controlled doses of helminths, such as human hookworm (termed hookworm therapy), is proposed as a treatment for many of the same diseases. Helminths induce immunoregulatory changes in their host which could decrease epithelial permeability, which is highlighted as a potential mechanism through which helminths treat disease. Despite this, the influence of a chronic helminth infection on epithelial permeability remains unclear. This study uses the chronically infecting intestinal helminth Heligmosomoides polygyrus to reveal alterations in the expression of intestinal tight junction proteins and epithelial permeability during the infection course. In the acute infection phase (1 week postinfection), an increase in intestinal epithelial permeability is observed. Consistent with this finding, jejunal claudin-2 is upregulated and tricellulin is downregulated. By contrast, in the chronic infection phase (6 weeks postinfection), colonic claudin-1 is upregulated and epithelial permeability decreases. Importantly, this study also investigates changes in epithelial permeability in a small human cohort experimentally challenged with the human hookworm, Necator americanus. It demonstrates a trend toward small intestinal permeability increasing in the acute infection phase (8 weeks postinfection), and colonic and whole gut permeability decreasing in the chronic infection phase (24 weeks postinfection), suggesting a conserved epithelial response between humans and mice. In summary, our findings demonstrate dynamic changes in epithelial permeability during a chronic helminth infection and provide another plausible mechanism by which chronic helminth infections could be utilized to treat disease.

Histopathological changes in the greenshell mussel, Perna canaliculus, in response to chronic thermal stress.

Climate change associated temperature challenges pose a serious threat to the marine environment. Elevations in average sea surface temperatures are occurring and increasing frequency of marine heatwaves resulting in mortalities of organisms are being reported. In recent years, marine farmers have reported summer mass mortality events of the New Zealand Greenshell mussel, Perna canaliculus, during the summer months; however, the etiological agents have yet to be determined. To elucidate the role of thermal stress, adult P. canaliculus were exposed to three chronic temperature treatments: a benign control of 17 °C and stressful elevations of 21 °C and 24 °C. Eight mussels per treatment were collected each month throughout a 14-month challenge period to identify and investigate histopathological differences among P. canaliculus populations exposed to the three temperatures. Histopathology revealed several significant deleterious alterations to tissues associated with temperature and exposure time. Increasing temperature and progression of time resulted in 1) an increase in the number of focal lipofuscin-ceroid aggregations, 2) an increase in focal hemocytosis, 3) an increase in the thickness of the sub-epithelial layer of the intestinal tract and 4) a decreased energy reserve cell (glycogen) coverage in the mantle. Prolonged exposure, irrespective of temperature, impacted gametogenesis, which was effectively arrested. Furthermore, increased levels of the heat shock protein 70 kDa (HSP 70) were seen in gill and gonad from thermally challenged mussels. The occurrence of the parasite Perkinsus olseni at month 5 in the 24 °C treatment, and month 7 at 21 °C was unexpected and may have exacerbated the fore-mentioned tissue conditions. Prolonged exposure to stable thermal conditions therefore appears to impact P. canaliculus, tissues with implications for broodstock captivity. Mussels experiencing elevated, temperatures of 21 and 24 °C demonstrated more rapid pathological signs. This research provides further insight into the complex host-pathogen-environment interactions for P. canaliculus in response to prolonged elevated temperature.

Skin Whole-Mount Immunofluorescent Staining Protocol, 3D Visualization, and Spatial Image Analysis.

The use of polychromatic immunofluorescent staining on whole-mount skin enables cell type characterization and aids in the delineation of the physiological and immunological strategies used by the skin to combat pathogens. Using whole-mount skin for polychromatic immunofluorescent staining removes the need for histological sectioning and enables the visualization of anatomical structures and immune cell types in three dimensions. Here we present a detailed protocol for immunostaining with fluorescence-conjugated primary antibodies in whole-mount skin to reveal structural landmarks and specific immune cell types using confocal laser scanning microscopy (CLSM) (Basic Protocol 1). The optimized staining panel reveals structural features such as blood vessels (CD31 antibody) and the lymphatic network (LYVE-1 antibody), in combination with MHCII antibodies for antigen-presenting cells (APCs), CD64 for macrophages and monocytes, CD103 for dendritic epidermal T cells (DETC), and CD326 for Langerhans cells (LC). Basic Protocol 2 describes image visualization pipelines using open-source software (ImageJ/FIJI), enabling four visualization options (z-projections, orthogonal views, 3D visualization, and animation). Basic Protocol 3 describes a quantitative analysis pipeline using CellProfiler to characterize the spatial relationship between cell types using mathematical indices such as Spatial Distribution Index (SDI), Neighborhood Frequency (NF), and Normalized Median Evenness (NME). These protocols will enable researchers to stain, record, analyze, and interpret data from whole-mount skin using commercially available reagents in a CLSM-equipped laboratory and freely available analysis software. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Immunofluorescent staining and imaging for whole-mount mouse skin Basic Protocol 2: File rendering and visualization using FIJI Basic Protocol 3: Spatial image analysis using CellProfiler.

A guideline for the appropriate recognition of shared resource laboratories in publication.

The issue of what level of contribution warrants authorship, determining a fair order of authors and when and whom to acknowledge in publications is often a cause of debate, and in some instances, has also been a focus of conflict at certain institutions. Shared resource laboratories (SRLs) play a fundamental role in supporting publications, and SRL staff scientists can contribute to numerous areas such as experimental design, sample preparation, data acquisition, data analysis and manuscript drafting and review. However, SRL staff scientists are often unfairly omitted from the author list. To avoid SRLs and SRL staff scientist contributions going unnoticed, the authors have formulated a set of guidelines to aid in the conceptualization and recognition of the technical and intellectual contributions of SRLs. As a better understanding of the role SRL staff scientists play in the achievement of the scientific lead's experimental aims will foster a positive feedback loop, where acknowledgements can lead to more support and funding for SRLs and more engaged SRL staff capable of supporting discoveries and technological innovations that underpin major advancements in the field of life sciences.

Implementing High Dimensional Reduction Analysis on Histocytometric Data.

In a previous protocol article, we demonstrated construction of a histocytometry pipeline that is capable of both segmenting highly aggregated cell populations and retaining the original intensity data range of the input microscopy images. In the protocol presented here, using the output from the aforementioned article, we demonstrate how to phenotype the data using the high dimensional reduction analysis technique optimized t-distributed stochastic neighbor embedding (opt-t-SNE) and compare it to traditional manual gating. Additionally, we present a protocol illustrating the advantage of the inclusion of cell junction/membrane markers for accurately segmenting highly aggregated cell populations in ilastik. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Phenotyping lymph node populations using manual gating Basic Protocol 2: Phenotyping lymph node populations using t-SNE dimensional reduction Support Protocol: ilastik segmentation using a pan marker.

Glycolipid-peptide conjugate vaccines elicit CD8+ T-cell responses and prevent breast cancer metastasis.

Objectives: Metastasis is the principal cause of breast cancer mortality. Vaccines targeting breast cancer antigens have yet to demonstrate clinical efficacy, and there remains an unmet need for safe and effective treatment to reduce the risk of metastasis, particularly for people with triple-negative breast cancer (TNBC). Certain glycolipids can act as vaccine adjuvants by specifically stimulating natural killer T (NKT) cells to provide a universal form of T-cell help. Methods: We designed and made a series of conjugate vaccines comprising a prodrug of the NKT cell-activating glycolipid α-galactosylceramide covalently linked to tumor-expressed peptides, and assessed these using E0771- and 4T1-based breast cancer models in vivo. We employed peptides from the model antigen ovalbumin and from clinically relevant breast cancer antigens HER2 and NY-ESO-1. Results: Glycolipid-peptide conjugate vaccines that activate NKT cells led to antigen-presenting cell activation, induced inflammatory cytokines, and, compared with peptide alone or admixed peptide and α-galactosylceramide, specifically enhanced CD8+ T-cell responses against tumor-associated peptides. Primary tumor growth was delayed by vaccination in all tumor models. Using 4T1-based cell lines expressing HER2 or NY-ESO-1, a single administration of the relevant conjugate vaccine prevented tumor colonisation of the lung following intravenous inoculation of tumor cells or spontaneous metastasis from breast, respectively. Conclusion: Glycolipid-peptide conjugate vaccines that activate NKT cells prevent lung metastasis in breast cancer models and warrant investigation as adjuvant therapies for high-risk breast cancer.

Intratumoural administration of an NKT cell agonist with CpG promotes NKT cell infiltration associated with an enhanced antitumour response and abscopal effect.

Intratumoural administration of unmethylated cytosine-phosphate-guanine motifs (CpG) to stimulate toll-like receptor (TLR)-9 has been shown to induce tumour regression in preclinical studies and some efficacy in the clinic. Because activated natural killer T (NKT) cells can cooperate with pattern-recognition via TLRs to improve adaptive immune responses, we assessed the impact of combining a repeated dosing regimen of intratumoural CpG with a single intratumoural dose of the NKT cell agonist α-galactosylceramide (α-GalCer). The combination was superior to CpG alone at inducing regression of established tumours in several murine tumour models, primarily mediated by CD8+ T cells. An antitumour effect on distant untreated tumours (abscopal effect) was reliant on sustained activity of NKT cells and was associated with infiltration of KLRG1+ NKT cells in tumours and draining lymph nodes at both injected and untreated distant sites. Cytometric analysis pointed to increased exposure to type I interferon (IFN) affecting many immune cell types in the tumour and lymphoid organs. Accordingly, antitumour activity was lost in animals in which dendritic cells (DCs) were incapable of signaling through the type I IFN receptor. Studies in conditional ablation models showed that conventional type 1 DCs and plasmacytoid DCs were required for the response. In tumour models where the combined treatment was less effective, the addition of tumour-antigen derived peptide, preferably conjugated to α-GalCer, significantly enhanced the antitumour response. The combination of TLR ligation, NKT cell agonism, and peptide delivery could therefore be adapted to induce responses to both known and unknown antigens.

Creation of High-Dimensional Reduction Analysis-Compatible Histocytometry Files from Images of Densely-Packed Cells and/or Variable Stain Intensity.

The power of high-dimensional reduction techniques using multiparameter images has been demonstrated across a variety of different publications. Recently, we published an end-to-end low-cost GUI-based protocol for performing histocytometric spatial analysis on images derived from the most common microscope image formats. However, this protocol is limited by the normalized marker intensity outputs and the difficulty in processing images of highly aggregated and/or exceptionally heterogenous cell populations. Here we present the basic protocols required to construct an advanced histocytometric data file using only freeware. This data file is compatible with images containing cell nuclei clusters that are difficult to segment, and results in histocytometry files retaining the original marker intensity values of the microscopic images they were derived from. This is especially useful in cells that are phenotyped based on relative marker expression levels. Histocytometry data files produced by these protocols are compatible with high-dimensional reduction analysis using marker intensity data, such as tSNEs. This methodology is showcased using stitched microscopic images of murine lymph nodes, complex organs with highly aggregated heterogenous cell populations, that are typically difficult to segment. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Image preprocessing and generation of nuclei marker probability maps Basic Protocol 2: Cell segmentation using ilastik-derived probability maps Basic Protocol 3: Generation of histocytometric .fcs files.

How to Build an Image-Processing Pipeline for Automating Multiparameter Histocytometry Analysis.

Until relatively recently, analysis of imaging data has been primarily quantitative and limited to 3-4 markers. The advancement of various technologies overcoming this marker limitation provided the capability of analyzing multiparameter imaging data down to the single cell level, termed histocytometry. Currently, most published end-to-end histocytometric analysis of imaging data is performed using expensive commercial programs or freely available analysis packages that require significant knowledge of programming languages for execution. Here we present a protocol that performs cell segmentation, phenotyping and spatial analysis, using software with easy-to-use GUIs (graphical user interfaces). These protocols allow the user to derive spatial and phenotypical data for the analysis of multiparameter microscopic images from most imaging platforms in a low-cost manner. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Cell Segmentation and generation of histocytometric .csv file Basic Protocol 2: Phenotyping of cell populations Basic Protocol 3: Spatial relationship analyses of phenotyped populations Support Protocol 1: Nuclei Segmentation Accuracy Test Support Protocol 2: Correcting y-axis Inversion of Histocytometry Data Relative to Original Image File.

A new method to localise and quantify oxidative stress in live juvenile mussels.

Stress and survival of the juvenile New Zealand green-lipped mussel, Perna canaliculus, is a poorly understood bottleneck in the ecological and economic performance of a significant aquaculture crop. This species was therefore selected as a model organism for the development of a new method to quantify oxidative stress in whole individuals. An in vivo ROS-activated stain (CellROX™) was administered to anaesthetised, translucent juveniles that were subsequently formaldehyde fixed and then visualised using confocal microscopy. Subsequent application of image analysis to quantifying ROS-positive tissue areas was successfully used to detect stress differences in juvenile mussels exposed to varying levels of emersion. This integrated method can be used to localise and quantify ROS production in individual translucent bivalve life stages (larval and juvenile), while relative stability following fixation greatly expands potential practical field applications. This article has an associated First Person interview with the first and third authors of the paper.

MR1-dependent immune surveillance of the skin contributes to pathogenesis and is a photobiological target of UV light therapy in a mouse model of atopic dermatitis.

BACKGROUND: Mucosal-associated invariant T (MAIT) cells are unconventional T cells which recognize microbial metabolites presented by the major histocompatibility complex class I-related molecule MR1. Although MAIT cells have been shown to reside in human and murine skin, their contribution to atopic dermatitis (AD), an inflammatory skin disease associated with barrier dysfunction and microbial translocation, has not yet been determined. METHODS: Genetic deletion of MR1 and topical treatment with inhibitory MR1 ligands, which result in the absence and functional inhibition of MAIT cells, respectively, were used to investigate the role of MR1-dependent immune surveillance in a MC903-driven murine model of AD. RESULTS: The absence or inhibition of MR1 arrested AD disease progression through the blockade of both eosinophil activation and recruitment of IL-4- and IL-13-producing cells. In addition, the therapeutic efficacy of phototherapy against MC903-driven AD could be increased with prior application of folate, which photodegrades into the inhibitory MR1 ligand 6-formylpterin. CONCLUSION: We identified MAIT cells as sentinels and mediators of cutaneous type 2 immunity. Their pathogenic activity can be inhibited by topical application or endogenous generation, via phototherapy, of inhibitory MR1 ligands.

High-Dimensional Image Analysis using Histocytometry.

Histocytometry is a technique for processing multiparameter microscopy images using computational approaches to identify and quantify cellular phenotypes. It allows for spatial analyses of cellular phenotypes in relation to each other and within defined spatial regions. The benefit of this technique over manual annotation and characterization of cells is a high degree of automation/throughput, significantly decreased user bias, and increased reproducibility. Recently, an increase in freely available software amenable to or deliberately designed for histocytometry has resulted in these complex analyses being available to a broader base of users who have amassed multi-component microscopic imaging data. This article provides an overview of a histocytometry pipeline, focusing on the strategic planning and software requirements to allow readers to perform cell segmentation, phenotyping, and spatial analyses to advance their research outputs. © 2021 Wiley Periodicals LLC.

Basophils promote barrier dysfunction and resolution in the atopic skin.

BACKGROUND: The type 2 cytokines IL-4 and IL-13 promote not only atopic dermatitis (AD) but also the resolution of inflammation. How type 2 cytokines participate in the resolution of AD is poorly known. OBJECTIVE: Our aim was to determine the mechanisms and cell types governing skin inflammation, barrier dysfunction, and resolution of inflammation in a model of AD. METHODS: Mice that exhibit expression of IL-4, IL-13, and MCPT8 or that could be depleted of basophils or eosinophils, be deficient in IL-4 or MHC class II molecules, or have basophils lacking macrophage colony-stimulating factor (M-CSF) were treated with calcipotriol (MC903) as an acute model of AD. Kinetics of the disease; keratinocyte differentiation; and leukocyte accumulation, phenotype, function, and cytokine production were measured by transepidermal water loss, histopathology, molecular biology, or unbiased analysis of spectral flow cytometry. RESULTS: In this model of AD, basophils were activated systemically and were the initial and main source of IL-4 in the skin. Basophils and IL-4 promoted epidermal hyperplasia and skin barrier dysfunction by acting on keratinocyte differentiation during inflammation. Basophils, IL-4, and basophil-derived M-CSF inhibited the accumulation of proinflammatory cells in the skin while promoting the expansion and function of proresolution M2-like macrophages and the expression of probarrier genes. Basophils kept their proresolution properties during AD resolution. CONCLUSION: Basophils can display both beneficial and detrimental type 2 functions simultaneously during atopic inflammation.

Dermal IRF4+ dendritic cells and monocytes license CD4+ T helper cells to distinct cytokine profiles.

Antigen (Ag)-presenting cells (APC) instruct CD4+ helper T (Th) cell responses, but it is unclear whether different APC subsets contribute uniquely in determining Th differentiation in pathogen-specific settings. Here, we use skin-relevant, fluorescently-labeled bacterial, helminth or fungal pathogens to track and characterize the APC populations that drive Th responses in vivo. All pathogens are taken up by a population of IRF4+ dermal migratory dendritic cells (migDC2) that similarly upregulate surface co-stimulatory molecules but express pathogen-specific cytokine and chemokine transcripts. Depletion of migDC2 reduces the amount of Ag in lymph node and the development of IFNγ, IL-4 and IL-17A responses without gain of other cytokine responses. Ag+ monocytes are an essential source of IL-12 for both innate and adaptive IFNγ production, and inhibit follicular Th cell development. Our results thus suggest that Th cell differentiation does not require specialized APC subsets, but is driven by inducible and pathogen-specific transcriptional programs in Ag+ migDC2 and monocytes.

The Gastrointestinal Helminth Heligmosomoides bakeri Suppresses Inflammation in a Model of Contact Hypersensitivity.

Helminths regulate host immune responses to ensure their own long-term survival. Numerous studies have demonstrated that these helminth-induced regulatory mechanisms can also limit host inflammatory responses in several disease models. We used the Heligmosomoides bakeri (Hb) infection model (also known as H. polygyrus or H. polygyrus bakeri in the literature) to test whether such immune regulation affects skin inflammatory responses induced by the model contact sensitiser dibutyl phthalate fluorescein isothiocynate (DBP-FITC). Skin lysates from DBP-FITC-sensitized, Hb-infected mice produced less neutrophil specific chemokines and had significantly reduced levels of skin thickening and cellular inflammatory responses in tissue and draining lymph nodes (LNs) compared to uninfected mice. Hb-induced suppression did not appear to be mediated by regulatory T cells, nor was it due to impaired dendritic cell (DC) activity. Mice cleared of infection remained unresponsive to DBP-FITC sensitization indicating that suppression was not via the secretion of Hb-derived short-lived regulatory molecules, although long-term effects on cells cannot be ruled out. Importantly, similar helminth-induced suppression of inflammation was also seen in the draining LN after intradermal injection of the ubiquitous allergen house dust mite (HDM). These findings demonstrate that Hb infection attenuates skin inflammatory responses by suppressing chemokine production and recruitment of innate cells. These findings further contribute to the growing body of evidence that helminth infection can modulate inflammatory and allergic responses via a number of mechanisms with potential to be exploited in therapeutic and preventative strategies in the future.

High-dimensional analysis of intestinal immune cells during helminth infection.

Single cell isolation from helminth-infected murine intestines has been notoriously difficult, due to the strong anti-parasite type 2 immune responses that drive mucus production, tissue remodeling and immune cell infiltration. Through the systematic optimization of a standard intestinal digestion protocol, we were able to successfully isolate millions of immune cells from the heavily infected duodenum. To validate that these cells gave an accurate representation of intestinal immune responses, we analyzed them using a high-dimensional spectral flow cytometry panel and confirmed our findings by confocal microscopy. Our cell isolation protocol and high-dimensional analysis allowed us to identify many known hallmarks of anti-parasite immune responses throughout the entire course of helminth infection and has the potential to accelerate single-cell discoveries of local helminth immune responses that have previously been unfeasible.

Parasitic worms known as helminths represent an important health problem in large parts of Africa, South America and Asia. Once their larvae enter the body, they head to the gut where they mature into adults and start laying eggs. In areas with poor sanitation, these may then get passed on to other individuals. To defend the body, the immune system sends large numbers of immune cells to the gut, but it usually struggles to eliminate the parasites. Without deworming medication, the infection can last for many years. Scientists study helminth infections in the laboratory by using worms that naturally infect mice. Understanding exactly how the immune system responds to the infection is essential to grasp why it fails to clear the worms. However, it is difficult to extract immune cells from an infected gut, as the infection creates strong local responses ­ such as an intense 'slime' production to try to flush out the worms. The standard procedure to obtain immune cells from the gut consists of three steps: collecting a gut segment and washing it, stripping away the surface layers with chemicals, and finally using enzymes to digest the tissues, which are then filtered to obtain individual cells. However, this protocol is not able to extract cells during infection. Ferrer-Font et al. therefore methodically refined every step of this method, and finally succeeded in obtaining millions of immune cells from infected guts. For the first time, these cells could then be studied and identified using a new technology called spectral flow cytometry. Over 40 immune cell types were followed throughout the course of infection, revealing that many 'first responders' immune cells were recruited to the gut early on, when the worms were still larvae. However, these cells disappeared once the worms developed into adults. These findings were confirmed by microscopy, which also showed that the first responder cells were found around the developing larvae, likely attacking them. When the adult worms developed, these cells were replaced by other immune cells, which also decreased the longer the worms were present in the gut. This new extraction process established by Ferrer-Font et al. can also be paired with other technologies that can, for example, reveal which genes are turned on in individual cells. This could help map out exactly how the body fights helminth infections, and how to improve this response. The method could also be useful to extract immune cells from the gut in other challenging scenarios, such food allergies or inflammatory bowel disorders.

Hookworms Evade Host Immunity by Secreting a Deoxyribonuclease to Degrade Neutrophil Extracellular Traps.

Hookworms cause a major neglected tropical disease, occurring after larvae penetrate the host skin. Neutrophils are phagocytes that kill large pathogens by releasing neutrophil extracellular traps (NETs), but whether they target hookworms during skin infection is unknown. Using a murine hookworm, Nippostrongylus brasiliensis, we observed neutrophils being rapidly recruited and deploying NETs around skin-penetrating larvae. Neutrophils depletion or NET inhibition altered larvae behavior and enhanced the number of adult worms following murine infection. Nevertheless, larvae were able to mitigate the effect of NETs by secreting a deoxyribonuclease (Nb-DNase II) to degrade the DNA backbone. Critically, neutrophils were able to kill larvae in vitro, which was enhanced by neutralizing Nb-DNase II. Homologs of Nb-DNase II are present in other nematodes, including the human hookworm, Necator americanus, which also evaded NETs in vitro. These findings highlight the importance of neutrophils in hookworm infection and a potential conserved mechanism of immune evasion.

Simultaneous Polychromatic Immunofluorescent Staining of Tissue Sections and Consecutive Imaging of up to Seven Parameters by Standard Confocal Microscopy.

Confocal microscopy has been an important imaging tool for life scientists for over 20 years. Early techniques focused on indirect staining processes that involved staining with an unconjugated primary antibody, followed by incubation with a secondary fluorescent antibody that would reveal and amplify the signal of the primary antibody. With more and more directly conjugated fluorescent primary antibodies becoming commercially available, staining with multiple fluorescent primary antibodies is now more frequent. To date, staining with up to three primary antibodies and a nuclear dye is widely practiced. Here, we describe an important improvement to the standard polychromatic immunofluorescent staining protocol that allows the simultaneous detection of seven fluorescent parameters using a standard confocal laser scanning microscope with four laser lines and four photomultiplier tubes. By incorporating recently available tandem dyes that emit in the blue and violet regions of the visible light spectrum (Brilliant Blue and Brilliant Violet), we were able to differentiate several additional fluorochromes simultaneously. Due to the added complexity of 7-color immunofluorescent imaging, we developed a clear methodology to optimize antibody concentrations and simple guidelines on how to identify and correct non-specific signals. These are detailed in the following protocol. © 2019 by John Wiley & Sons, Inc. Basic Protocol: 7-Color immunofluorescent staining protocol using directly conjugated antibodies Support Protocol 1: Antibody titration protocol Support Protocol 2: Spillover optimization protocol.