Journal of Biological Methods (JBM)
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163 research outputs found
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A simple and robust cell-based assay for the discovery of novel cytokinesis inhibitors
No full textCytokinesis is the last step of mitotic cell division that separates the cytoplasm of dividing cells. Small molecule inhibitors targeting either the elements of the regulatory pathways controlling cytokinesis, or the terminal effectors have been of interest as potential drug candidates for the treatment of various diseases. Here we present a detailed protocol for a cell-based cytokinesis assay that can be used for the discovery of novel cytokinesis inhibitors. The assay is performed in a 96-well plate format in 48 h. Living cells, nuclei and nuclei of dead cells are identified by a single staining step using three fluorescent dyes, followed by rapid live cell imaging. The primary signal is the nuclei-to-cell ratio (NCR). In the presence of cytokinesis inhibitors, this ratio increases over time, as the ratio of multinucleated cells increases in the population. The ratio of dead nuclei to total nuclei provides a simultaneous measure of cytotoxicity. A screening window coefficient (Z`) of 0.65 indicates that the assay is suitable for screening purposes, as the positive and negative controls are well-separated. EC50 values can be reliably determined in a single 96-well plate by using only six different compound concentrations, enabling the testing of 4 compounds per plate. An excellent test-retest reliability (R2 = 0.998) was found for EC50 values covering a ~1500-fold range of potencies. Established small molecule inhibitors of cytokinesis operating via direct action on actin dynamics or nonmuscle myosin II are used to demonstrate the robustness, simplicity and flexibility of the assay
Generation of bone marrow chimeras using X-ray irradiation: comparison to cesium irradiation and use in immunotherapy
No full textBone marrow chimeras represent a key tool employed to understand biological contributions stemming from the hematopoietic versus the stromal compartment. In most institutions, cesium irradiators are used to lethally irradiate recipient animals prior to the injection of donor bone marrow. Cesium irradiators, however, have significant liabilities—including concerns around domestic security. Recently, X-ray irradiators have been implemented as a potential alternative to cesium sources. Only a small number of publications in the literature have attempted to compare these two modalities and, in most cases, the emphasis was on irradiation of human blood productions. We were able to find only a single study that directly compared X-ray and cesium technologies in the generation of murine bone marrow chimeras, a standard laboratory practice. This study focused on chimerism in the blood of recipient animals. In the present study, we begin by comparing cesium and X-ray based sources for irradiation, then transition to using X-ray-based systems for immunology models with an emphasis on immunotherapy of cancer in immunocompetent mouse models—specifically evaluating chimerism in the blood, spleen, and tumor microenvironment. While our data demonstrate that the two platforms are functionally comparable and suggest that X-ray based technology is a suitable alternative to cesium sources. We also highlight a difference in chimerism between the peripheral (blood, spleen) and tumor compartments that is observed using both technologies. While the overall degree of chimerism in the peripheral tissues is very high, the degree of chimerism in the tumor is cell type specific with T and NK cells showing lower chimerism than other cell types
Optimized methodology for product recovery following emulsion PCR: applications for amplification of aptamer libraries and other complex templates
No full textBias and background issues make efficient amplification of complex template mixes such as aptamer and genomic DNA libraries via conventional PCR methods difficult; emulsion PCR is being increasingly used in such scenarios to circumvent these problems. However, before products generated via emulsion PCR can be used in downstream workflows, they need to be recovered from the water-in-oil emulsion. Often, emulsions are broken following amplification using volatile organic solvents, and product is subsequently isolated via precipitation. Unfortunately, the use of such solvents requires the implementation of special environmental controls, and the yield and purity of DNA isolated by precipitation can be highly variable. Here, we describe the optimization of a simple protocol which can be used to recover products following emulsion PCR using a 2-butanol extraction and subsequent DNA isolation via a commercially available clean-up kit. This protocol avoids the use of volatile solvents and precipitation steps, and we demonstrate that it can be used to reliably recover DNA from water-in-oil emulsions with efficiencies as high as 90%. Furthermore, we illustrate the practical applicability of this protocol by demonstrating how it can be implemented to recover a complex random aptamer library following amplification via emulsion PCR
Highly efficient induced pluripotent stem cell reprogramming of cryopreserved lymphoblastoid cell lines
No full textTissue culture based in-vitro experimental modeling of human inherited disorders provides insight into the cellular and molecular mechanisms involved and the underlying genetic component influencing the disease phenotype. The breakthrough development of induced pluripotent stem cell (iPSC) technology represents a quantum leap in experimental modeling of human diseases, providing investigators with a self-renewing and thus unlimited source of pluripotent cells for targeted differentiation into functionally relevant disease specific tissue/cell types. The existing rich bio-resource of Epstein-Barr virus (EBV) immortalized lymphoblastoid cell line (LCL) repositories generated from a wide array of patients in genetic and epidemiological studies worldwide, many of them with extensive genotypic, genomic and phenotypic data already existing, provides a great opportunity to reprogram iPSCs from any of these LCL donors in the context of their own genetic identity for disease modeling and disease gene identification. However, due to the low reprogramming efficiency and poor success rate of LCL to iPSC reprogramming, these LCL resources remain severely underused for this purpose. Here, we detailed step-by-step instructions to perform our highly efficient LCL-to-iPSC reprogramming protocol using EBNA1/OriP episomal plasmids encoding pluripotency transcription factors (i.e., OCT3/4, SOX2, KLF4, L-MYC, and LIN28), mouse p53DD (p53 carboxy-terminal dominant-negative fragment) and commercially available reprogramming media. We achieved a consistently high reprogramming efficiency and 100% success rate (> 200 reprogrammed iPSC lines) using this protocol
A simple method for long-term vital-staining of ciliated epidermal cells in aquatic larvae
No full textObserving the process of growth and differentiation of tissues and organs is of crucial importance for the understanding of the evolution of organs in animals. Unfortunately, it is notoriously difficult to continuously monitor developmental processes due to the extended time they take. Long-term labeling of the tissues of interest represents a promising alternative to raise these pivotal data. In the case of the prototroch, a band of ciliated cells typical of marine, planktotrophic trochophora larvae, we were able to apply a long-term fluorescent vital-staining to the prototroch cells that remains detectable throughout further larval life. We were able to stain ciliated cells of planktonic larvae from different spiralian clades by using long-chain dialkylcarbocyanine dyes that are detectable in different fluorescent emission spectra in combination with a non-ionic surfactant. The larvae survived and developed normally, their ciliated cells retaining the originally applied fluorescent labels. Combined with additional fluorescent staining of the larvae after fixation, we provide an easy, versatile, and broadly applicable method to investigate the processes of the differentiation of epidermal organs in various aquatic larvae
A streamlined CRISPR/Cas9 approach for fast genome editing in Toxoplasma gondii and Besnoitia besnoiti
No full textToxoplasma gondii (T. gondii) and Besnoitia besnoiti (B. besnoiti) are closely related coccidian parasites belonging to the phylum Apicomplexa, which comprises many other important pathogens of humans and livestock. T. gondii is considered a model organism for studying the cell biology of Apicomplexa mainly due to the ease of propagation in diverse host cells and the availability of a wide range of genetic tools. Conversely, B. besnoiti in vitro culture systems currently exist only for the acute phase of infection, and genetic manipulation has proven much more challenging. In recent years, the targeted editing of chromosomal DNA by the programmable CRISPR-associated (Cas)9 enzyme has greatly improved the scope and accuracy of genetic manipulation in T. gondii and related parasites but is still lagging in B. besnoiti. The CRISPR/Cas9 technology enables the introduction of single point and insertion/deletion mutations, precise integration of in-frame epitope tags, and deletions of genes at reduced time and cost compared to previous methods. Current protocols for CRISPR-mediated genome editing in T. gondii rely on either constitutive or transient expression of Cas9 as well as target specific sgRNAs encoded separately or together on transfected plasmid vectors. Constitutively expressed Cas9 carries the risk of toxicity, whilst the transient approach is laborious and error-prone. Here we present a protocol for plasmid vector-independent genome-editing using chemically synthesized and modified sgRNAs. This protocol allows for rapid, efficient, and cost-effective generation of mutant cell lines of T. gondii and B. besnoiti
Comparing NGS and NanoString platforms in peripheral blood mononuclear cell transcriptome profiling for advanced heart failure biomarker development
No full textIn preparation to create a clinical assay that predicts 1-year survival status of advanced heart failure (AdHF) patients before surgical/interventional therapies and to select the appropriate clinical assay platform for the future assay, we compared the properties of next generation sequencing (NGS) used in the gene discovery phase to the NanoString platform used in the clinical assay development phase. In 25 AdHF patients in a tertiary academic medical center from 2015 to 2016, PBMC samples were collected and aliquoted for NGS RNA whole transcriptome sequencing and compared to 770 genes represented on NanoString’s PanCancer IO 360 Gene Expression research panel. Prior to statistical analysis, NanoString and NGS expression values were log transformed. We computed Pearson correlation coefficients for each sample, comparing gene expression values between NanoString and NGS across the set of matched genes and for each of the matched genes across the set of samples. Genes were grouped by average NGS expression, and the NanoString-NGS correlation for each group was computed. Out of 770 genes from the NanoString panel, 734 overlapped between both platforms and showed high intrasample correlation. Within an individual sample, there was an expression-level dependent correlation between both platforms. The low- vs. intermediate/high-expression groups showed NGS average correlation 0.21 vs. 0.58–0.68, respectively, and NanoString average correlation 0.07–0.34 vs. 0.59–0.70, respectively. NanoString demonstrated high reproducibility (R2 > 0.99 for 100 ng input), sensitivity (probe counts between 100 and 500 detected and quantified), and robustness (similar gene signature scores across different RNA input concentrations, cartridges, and outcomes). Data from NGS and NanoString were highly correlated. These platforms play a meaningful, complementary role in the biomarker development process
An ex vivo skin model to probe modulation of local cutaneous arachidonic acid inflammation pathway
No full textThere is a need for inexpensive and reliable means to determine the modulation of cutaneous inflammation. The method outlined in this article draws together a number of scientific techniques and makes use of generally unwanted biological tissues as a means of determining skin inflammation ex vivo, and focuses on probing aspects of the arachidonic acid inflammation pathway. Freshly excised skin contains elevated levels of short-lived inducible cyclooxygenase-2 (COX-2) and, under viable conditions, COX-2 and its eicosanoid products will continue to be produced until tissue necrosis, providing a window of time in which relative levels can be probed to determine exacerbation due to an upregulating factor or downregulation due the presence of an agent exerting anti-inflammatory activity. Ex vivo porcine skin, mounted in Franz diffusion cells, is dosed topically with the xenobiotic challenge and then techniques such as Western blotting and immunohistochemistry can then be used to probe relative COX-2 levels on a semi-quantitative or qualitative level. Enzyme-linked immunosorbent assay or LCMS can be used to determine relative prostaglandin E-2 (PGE-2) levels. Thus far, the technique has been used to examine the effects of topically applied anti-inflammatories (betamethasone, ibuprofen, ketoprofen and methotrexate), natural products (fish oil, Devil’s claw extract and pomegranate rind extract) and drug delivery vehicle (polyNIPAM nanogels). Topically applied xenobiotics that modulate factors such as COX-2 and PGE-2 must penetrate the intact skin, and this provides direct evidence of overcoming the "barrier function" of the stratum corneum in order to target the viable epidermis in sufficient levels to be able to elicit such effects. This system has particular potential as a pre-clinical screening tool for those working on the development of topical delivery systems, and has the additional advantage of being in line with 3 Rs philosophy
A system for the high-throughput analysis of acute thermal avoidance and adaptation in C. elegans
No full textNociception and its plasticity are essential biological processes controlling adaptive behavioral responses in animals. These processes are also linked to different pain conditions in human and have received considerable attention, notably via studies in rodent models and the use of heat-evoked withdrawal behavior assays as a readout of unpleasant experience. More recently, invertebrates have also emerged as useful complementary models, with their own set of advantages, including their amenability to genetic manipulations, the accessibility and relative simplicity of their nervous system and ethical concerns linked to animal suffering. Like humans, the nematode Caenorhabditis elegans (C. elegans) can detect noxious heat and produce avoidance responses such as reversals. Here, we present a methodology suitable for the high-throughput analysis of C. elegans heat-evoked reversals and the adaptation to repeated stimuli. We introduce two platforms: the INFERNO (for infrared-evoked reversal analysis platform), allowing the quantification of the thermal sensitivity in a petri dish containing a large population (> 100 animals), and the ThermINATOR (for thermal adaptation multiplexed induction platform), allowing the mass-adaptation of up to 18 worm populations at the same time. We show that wild type animals progressively desensitize in response to repeated noxious heat pulses. Furthermore, analyzing the phenotype of mutant animals, we show that the mechanisms underlying baseline sensitivity and adaptation, respectively, are supported by genetically separable molecular pathways. In conclusion, the presented method enables the high-throughput evaluation of thermal avoidance in C. elegans and will contribute to accelerate studies in the field with this invertebrate model
A simple method for short-term maintenance of neonatal mice without foster mothers
No full textMice are typically weaned from their mother between 21 and 28 days of age, or at 10 grams of body weight. However, some biochemical experiments need to be done before the weaning days, and the mother might cannibalize or ignore those manipulated pups. Here, we provide a detailed protocol for maintenance of neonatal mice without the presence of their mothers for biomedical research. The basic instinct of neonate mice to hide under covers is harnessed for their survival in a mother-free environment. When covers are soaked with milk and the only targets for hiding, the neonates would acquire their nutrients at least in an involuntary fashion. The protocol is simple and can be used for neonatal rodent studies for short periods of times, and assures the accuracy of the biomedical experiments if survival rate of neonates is critical