High-content Cell Imaging Research Articles

Expanding TheCellVision.org: a central repository for visualizing and mining high-content cell imaging projects.

We previously constructed TheCellVision.org, a central repository for visualizing and mining data from yeast high-content imaging projects. At its inception, TheCellVision.org housed two high-content screening (HCS) projects providing genome-scale protein abundance and localization information for the budding yeast Saccharomyces cerevisiae, as well as a comprehensive analysis of the morphology of its endocytic compartments upon systematic genetic perturbation of each yeast gene. Here, we report on the expansion of TheCellVision.org by the addition of two new HCS projects and the incorporation of new global functionalities. Specifically, TheCellVision.org now hosts images from the Cell Cycle Omics project, which describes genome-scale cell cycle-resolved dynamics in protein localization, protein concentration, gene expression, and translational efficiency in budding yeast. Moreover, it hosts PIFiA, a computational tool for image-based predictions of protein functional annotations. Across all its projects, TheCellVision.org now houses >800,000 microscopy images along with computational tools for exploring both the images and their associated datasets. Together with the newly added global functionalities, which include the ability to query genes in any of the hosted projects using either yeast or human gene names, TheCellVision.org provides an expanding resource for single-cell eukaryotic biology.

Revealing the “Yin-Jing” mystery veil of Platycodon grandiflorum by potentiating therapeutic effects and lung-oriented guidance property

Ethnic pharmacological relevance“Yin-Jing” medicine (YJM) has been widely used by both ancient and modern Chinese medicine practitioners during long-term clinical practice. However, it remains unclear how to best guide other medicines to the targeted organs in a traditional Chinese medicine (TCM) prescription. Here, in an attempt to explain the scientific connotation of the YJM property (YJMP) attributed to a basic TCM theory, Platycodon grandiflorum (PG) was chosen as a case study to reveal the mystery of YJMP theory. Aim of the studyThe main purpose of this study is to employ modern chemical and molecular biology methods to confirm the “Yin-Jing” effect of PG, and further clarify its material basis and related possible mechanism. Materials and methodsThe ammonia-induced lung injury rat model was utilized to determine the optimal dosage of traditional prescription Hui Yan Zhu Yu decoction (HYZYD) using Wright Giemsa staining, HE staining, Masson staining, and TUNEL analysis. With the same way, PG was confirmed to have potentiating therapeutic effect (PTE) by comparison with HYZYD and [HYZYD-PG]. TMT proteomics was used to reveal the “Yin-Jing” mechanism of action. Western blot assay (WB) was employed for verification of differentially expressed proteins. Additionally, four non-crossing fragmentations (Fr. A-D) were characterized by RPLC/SEC-ELSD and HILIC-ESI–-Q-OT-IT-MS techniques. The PTE and guidance property assays were utilized to evaluate “Yin-Jing” functions by a compatible combination of hydroxysafflor yellow A (HYA) using qPCR, FCM, WB, HPLC, high content cell imaging (HCI) and high-resolution live-cell imaging (HRLCI) techniques. ResultsThe HYZYD-M (medium dose group) significantly improved the lung injury level in a pneumonia model of rats. PG enhanced the therapeutic effect of HYZYD ascribed to Yin-Jing PTE functions. TMT proteomics revealed a category of differentially expressed proteins ascribed to Golgi-ER between HYZYD and [HYZYD-PG]. Fr. C (i.e., saponins) and Fr. D (i.e., lipids) were determined as therapeutic fragmentations via the LPS-induced A549 cell injury model; however, Fr. B (fructooligosaccharides and small Mw fructans) had no therapeutic effect. Further compatibility PTE assays confirmed Fr. B significantly improved efficiency by a combination of HYA. The guidance assays showed Fr. B could significantly increase the uptake and distribution of HYA into lung cells and tissues. HCI assays showed that Fr. B increased uptake of HYA accompanied by significant activation of Golgi-ER. Unlike Fr. B, HRLCI showed that Fr. A, C and D were not only unobvious activations of Golgi-ER but also insignificant facilitation of colocalizations between HYA and Golgi-ER. ConclusionsFr. B is believed to be a key YJMP material basis of PG attributed to Yin-Jing PTE with characteristic of lung-oriented guidance property, whereas another abound Fr. C was determined to have synergistic effects rather than Yin-Jing material basis.

MAC-Seq: Coupling Low-Cost, High-Throughput RNA-Seq with Image-Based Phenotypic Screening in 2D and 3D Cell Models.

Transcriptomic profiling has fundamentally influenced our understanding of cancer pathophysiology and response to therapeutic intervention and has become a relatively routine approach. However, standard protocols are usually low-throughput, single-plex assays and costs are still quite prohibitive. With the evolving complexity of in vitro cell model systems, there is a need for resource-efficient high-throughput approaches that can support detailed time-course analytics, accommodate limited sample availability, and provide the capacity to correlate phenotype to genotype at scale. MAC-seq (multiplexed analysis of cells) is a low-cost, ultrahigh-throughput RNA-seq workflow in plate format to measure cell perturbations and is compatible with high-throughput imaging. Here we describe the steps to perform MAC-seq in 384-well format and apply it to 2D and 3D cell cultures. On average, our experimental conditions identified over ten thousand expressed genes per well when sequenced to a depth of one million reads. We discuss technical aspects, make suggestions on experimental design, and document critical operational procedures. Our protocol highlights the potential to couple MAC-seq with high-throughput screening applications including cell phenotyping using high-content cell imaging.

Morphological profiling of human T and NK lymphocytes by high-content cell imaging.

The immunological synapse is a complex structure that decodes stimulatory signals into adapted lymphocyte responses. It is a unique window to monitor lymphocyte activity because of development of systematic quantitative approaches. Here we demonstrate the applicability of high-content imaging to human T and natural killer (NK) cells and develop a pipeline for unbiased analysis of high-definition morphological profiles. Our approach reveals how distinct facets of actin cytoskeleton remodeling shape immunological synapse architecture and affect lytic granule positioning. Morphological profiling of CD8+ Tcells from immunodeficient individuals allows discrimination of the roles of the ARP2/3 subunit ARPC1B and the ARP2/3 activator Wiskott-Aldrich syndrome protein (WASP) in immunological synapse assembly. Single-cell analysis further identifies uncoupling of lytic granules and F-actin radial distribution in ARPC1B-deficient lymphocytes. Our study provides a foundation for development of morphological profiling as a scalable approach to monitor primary lymphocyte responsiveness and to identify complex aspects of lymphocyte micro-architecture.

TheCellVision.org: A Database for Visualizing and Mining High-Content Cell Imaging Projects.

Advances in genome engineering and high throughput imaging technologies have enabled genome-scale screens of single cells for a variety of phenotypes, including subcellular morphology and protein localization. We constructed TheCellVision.org, a freely available and web-accessible image visualization and data browsing tool that serves as a central repository for fluorescence microscopy images and associated quantitative data produced by high-content screening experiments. Currently, TheCellVision.org hosts ∼575,590 images and associated analysis results from two published high-content screening (HCS) projects focused on the budding yeast Saccharomyces cerevisiae. TheCellVision.org allows users to access, visualize and explore fluorescence microscopy images, and to search, compare, and extract data related to subcellular compartment morphology, protein abundance, and localization. Each dataset can be queried independently or as part of a search across multiple datasets using the advanced search option. The website also hosts computational tools associated with the available datasets, which can be applied to other projects and cell systems, a feature we demonstrate using published images of mammalian cells. Providing access to HCS data through websites such as TheCelllVision.org enables new discovery and independent re-analyses of imaging data.

Siwu Paste protects bone marrow hematopoietic function in rats with blood deficiency syndrome by regulating TLR4/NF-κB/NLRP3 signaling pathway

Ethnopharmacological relevanceSiwu Paste (SWP) was recorded in the first national Pharmacopoeia of China, “Tai Ping Hui Min He Ji Ju Fang”, it showed excellent effects in regulating all syndromes relevant to blood.Aim of the study: This study aimed to investigate the protective effects of Siwu Paste (SWP) on bone marrow hematopoietic by using rats’ model with blood deficiency syndrome induced by chemotherapy. Materials and methodsAnimal model with blood deficiency syndrome was successfully established by evaluating their peripheral blood cell level and erythrocyte membrane energy metabolism enzyme activity. Serum hematopoietic cytokine levels were detected by using Enzyme-linked immunosorbent assay (ELISA). Hematoxylin-Eosin (HE) staining method was used to observe the pathological morphology of femur bone marrow, and the viability of BMSC was detected by Cell Counting Kit (CCK-8). Furthermore, the expression of toll-like receptor 4 (TLR4), nuclear transcription factor kB (NF-κB), and NOD-like receptor protein 3 (NLRP3) protein in femur bone marrow were detected by using Western-blotting and High-content cell imaging analysis system (HCA). ResultsObtained results showed that SWP could significantly improve the status of anemia, regulate the expressions of serum hematopoietic cytokines, and protect bone marrow hematopoietic cells. Furthermore, the expressions of TLR4, NF-κB, and NLRP3 protein were inhibited in bone marrow hematopoietic cells. ConclusionsSiwu Paste (SWP) could recover the bone marrow hematopoietic functions in rats with blood deficiency syndrome. The therapeutic mechanism may be related to the regulation of serum hematopoietic cytokines, and inhibition of TLR4/NF-κB/NLRP3 signaling pathway.

Streamlined selection of cancer antigens for vaccine development through integrative multi-omics and high-content cell imaging

Identification of tumor antigens that induce cytotoxic T lymphocytes (CTLs) is crucial for cancer-vaccine development. Despite their predictive ability, current algorithmic approaches and human leukocyte antigen (HLA)-peptidomic analysis allow limited selectivity. Here, we optimized a method to rapidly screen and identify highly immunogenic epitopes that trigger CTL responses. We used a combined application of this method involving immune-specific signature analysis and HLA-associated peptidomics using samples from six patients with triple-negative breast cancer (TNBC) in order to select immunogenic HLA epitopes for in vitro testing. Additionally, we applied high-throughput imaging at the single-cell level in order to confirm the immunoreactivity of the selected peptides. The results indicated that this method enabled identification of promising CTL peptides capable of inducing antitumor immunity. This platform combining high-resolution computational analysis, HLA-peptidomics, and high-throughput immunogenicity testing allowed rapid and robust identification of highly immunogenic epitopes and represents a powerful technique for cancer-vaccine development.

Effects of ELK-1/JNK/c-Fos on apoptosis of rat hippocampal neurons cultured in vitro with Zuogui Jiangtang Jieyu Formula in simulated diabetes mellitus complicated with depression

To study the effects of Zuogui Jiangtang Jieyu Formula (ZGJTJYF, the Chinese Medicine) on hippocampal neuron apoptosis in diabetes mellitus complicated with depression (DD). The primary cultured hippocampal neurons were treated with high glucose (150 mmol/L) and corticosterone (200 micromol/L) to establish the cell model of DD in vitro. The cultured hippocampal neurons were randomly divided into five groups: blank serum group, normal group, Zuogui Jiangtang Jieyu recipe drug-containing serum group, positive drug (metformin + fluoxetine) drug-containing serum group and model group (three compound holes in each group). The model group and the normal group were given the same amount of culture medium, and the other groups were given the corresponding serum with 10% volume fraction for 18 hours. Hoechst staining, high content cell imaging and RT-PCR were used to detect the apoptosis of hippocampal neurons and the expressions of apoptosis-related ETS-like 1 transcription factor(ELK-1), C-Jun N-terminal kinase(JNK) and c-Fos proteins and genes. Compared with the blank group, the apoptotic number of hippocampal neurons in the model group was increased significantly, and the expression levels of ELK-1, JNK and c-Fos were increased significantly (P＜0.05). Compared with the model group, the local bright spots of hippocampal neurons in the Zuogui Jiangtang Jieyu recipe-containing serum group and the positive drug-containing serum group were decreased significantly, and the number of apoptotic cells was decreased significantly. The expressions of JNK, c-fos protein and mRNA were down-regulated significantly (P＜ 0.05), and the neural network and dendritic junction were improved significantly. Zuo Gui Jiang Tang Jie Yu Formula can reverse the expressions of ELK-1, JNK and c-Fos signals in hippocampal neurons under DD environment and play an anti-apoptotic effect.

Application of high-content cell imaging system in cell proliferation detection

Application of high-content cell imaging system in cell proliferation detection

Naturally produced opsonizing antibodies restrict the survival of Mycobacterium tuberculosis in human macrophages by augmenting phagosome maturation.

This study investigated the hypothesis that serum antibodies against Mycobacterium tuberculosis present in naturally infected healthy subjects of a tuberculosis (TB) endemic area could create and/or sustain the latent form of infection. All five apparently healthy Indian donors showed high titres of serum antibodies against M. tuberculosis cell membrane antigens, including lipoarabinomannan and alpha crystallin. Uptake and killing of bacilli by the donor macrophages was significantly enhanced following their opsonization with antibody-rich, heat-inactivated autologous sera. However, the capability to opsonize was apparent for antibodies against some and not other antigens. High-content cell imaging of infected macrophages revealed significantly enhanced colocalization of the phagosome maturation marker LAMP-1, though not of calmodulin, with antibody-opsonized compared with unopsonized M. tuberculosis. Key enablers of macrophage microbicidal action—proinflammatory cytokines (IFN-γ and IL-6), phagosome acidification, inducible NO synthase and nitric oxide—were also significantly enhanced following antibody opsonization. Interestingly, heat-killed M. tuberculosis also elevated these mediators to the levels comparable to, if not higher than, opsonized M. tuberculosis. Results of the study support the emerging view that an efficacious vaccine against TB should, apart from targeting cell-mediated immunity, also generate ‘protective’ antibodies.

High-Content Assay Multiplexing for Toxicity Screening in Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Hepatocytes.

Cell-based high-content screening (HCS) assays have become an increasingly attractive alternative to traditional in vitro and in vivo testing in pharmaceutical drug development and toxicological safety assessment. The time- and cost-effectiveness of HCS assays, combined with the organotypic nature of human induced pluripotent stem cell (iPSC)-derived cells, open new opportunities to employ physiologically relevant in vitro model systems to improve screening for potential chemical hazards. In this study, we used two human iPSC types, cardiomyocytes and hepatocytes, to test various high-content and molecular assay combinations for their applicability in a multiparametric screening format. Effects on cardiomyocyte beat frequency were characterized by calcium flux measurements for up to 90 min. Subsequent correlation with intracellular cAMP levels was used to determine if the effects on cardiac physiology were G-protein-coupled receptor dependent. In addition, we utilized high-content cell imaging to simultaneously determine cell viability, mitochondrial integrity, and reactive oxygen species (ROS) formation in both cell types. Kinetic analysis indicated that ROS formation is best detectable 30 min following initial treatment, whereas cytotoxic effects were most stable after 24 h. For hepatocytes, high-content imaging was also used to evaluate cytotoxicity and cytoskeletal integrity, as well as mitochondrial integrity and the potential for lipid accumulation. Lipid accumulation, a marker for hepatic steatosis, was most reliably detected 48 h following treatment with test compounds. Overall, our results demonstrate how a compendium of assays can be utilized for quantitative screening of chemical effects in iPSC cardiomyocytes and hepatocytes and enable rapid and cost-efficient multidimensional biological profiling of toxicity.

Tracking transcriptional activities with high-content epifluorescent imaging

High-content cell imaging based on fluorescent protein reporters has recently been used to track the transcriptional activities of multiple genes under different external stimuli for extended periods. This technology enhances our ability to discover treatment-induced regulatory mechanisms, temporally order their onsets and recognize their relationships. To fully realize these possibilities and explore their potential in biological and pharmaceutical applications, we introduce a new data processing procedure to extract information about the dynamics of cell processes based on this technology. The proposed procedure contains two parts: (1) image processing, where the fluorescent images are processed to identify individual cells and allow their transcriptional activity levels to be quantified; and (2) data representation, where the extracted time course data are summarized and represented in a way that facilitates efficient evaluation. Experiments show that the proposed procedure achieves fast and robust image segmentation with sufficient accuracy. The extracted cellular dynamics are highly reproducible and sensitive enough to detect subtle activity differences and identify mechanisms responding to selected perturbations. This method should be able to help biologists identify the alterations of cellular mechanisms that allow drug candidates to change cell behavior and thereby improve the efficiency of drug discovery and treatment design.

Exploiting the determinants of stochastic gene expression in Saccharomyces cerevisiae for genome-wide prediction of expression noise

Gene regulation is a process with many steps allowing for stochastic biochemical reactions, which leads to expression noise-i.e., the cell-to-cell stochastic fluctuation in protein abundance. Such expression noise can give rise to drastically diverse phenotypes, even within isogenic cell populations. Although numerous biophysical approaches had been proposed to model the origin and propagation of expression noise in biological networks, these models essentially characterize the innate stochastic dynamics in gene regulation in a mechanistic way. In this work, by investigating expression noise in the context of yeast cellular networks, we place the biophysical formulism onto solid genetic ground. At the sequence level, we show that extremely noisy genes are highly conserved in their coding sequences. At the level of cellular networks where natural selection is manifested by the topological constraints, we show that genes with varying expression noise are modularly organized in the protein interaction network and are positioned orderly in the gene regulatory network. We demonstrate that these topological constraints are highly predictive of stochastic gene expression, with which we were able to confidently predict stochastic expression for more than 2,000 yeast genes whose expression noise was previously not known. We validated the predictions by high-content cell imaging. Our approach makes feasible genome-wide prediction of stochastic gene expression, and such predictability in turn suggests that expression noise is an evolvable genetic trait.