Journal of biological methods最新文献

OpenArray is one of the most high-throughput qPCR platforms available but its efficiency can be limited by sample preparation methods that are slow and costly. To optimize the sample workflow for high-throughput qPCR processing by OpenArray, small-scale sample preparation methods were compared for compatibility with this system to build confidence in a method that maintains quality and accuracy while using less starting material and saving time and money. This study is the first to show that the Cells-to-CT kit can be used to prepare samples within the dynamic range of OpenArray directly from cultured cells in a single well of a 96-well plate when used together with a cDNA preamplification PCR step. Use of Cells-to-CT produced results of similar quality and accuracy to that of a preparation method using purified RNA in less than half the sample preparation time. While Cells-to-CT samples also exhibited slightly increased variance, which affects the ability of OpenArray to distinguish small differences in gene expression, overall gene expression mean results correlated well between small-scale methods. This work demonstrates that Cells-to-CT with preamplification can be used to reliably prepare samples for OpenArray analysis while saving time, money, and starting material.

We measured anomalous diffusion in human prostate cancer cells which were transfected with the Alexa633 fluorescent RNA probe and co-transfected with enhanced green fluorescent protein-labeled argonaute2 protein by laser scanning microscopy. The image analysis arose from diffusion based on a "two-level system". A trap was an interaction site where the diffusive motion was slowed down. Anomalous subdiffusive spreading occurred at cellular traps. The cellular traps were not immobile. We showed how the novel analysis method of imaging data resulted in new information about the number of traps in the crowded and heterogeneous environment of a single human prostate cancer cell. The imaging data were consistent with and explained by our modern ideas of anomalous diffusion of mixed origins in live cells. Our original research presented in this study is significant as we obtained a complex diffusion mechanism in live single cells.

An organoid is a three-dimensional multicellular structure that shows realistic micro-anatomy of an organ. This in vitro model mimics the in vivo environment, architecture and multi-lineage differentiation of the original organs and allows to answer many interesting biological questions. For these reasons, they are widely used in stem cell, regenerative medicine, toxicology, pharmacology, and host-microbe interactions research. In order to study organoids, microscopy is very useful: It is possible to make three-dimensional reconstruction of serial sections but it is time consuming and error-prone. Here we propose an alternative solution: Tissue clearing reduces the dispersion of light because it homogenizes the refractive index of the tissue, allowing sample observation throughout its thickness. We have compared different clearing techniques on mouse intestinal organoids using different acquisition methods.

Toxoplasma 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 and cost-effective generation of mutant cell lines of T. gondii and B. besnoiti.

Current methodologies to measure apoptotic and necrotic cell death using flow cytometry do not adequately differentiate between the two. Here, we describe a flow cytometry methodology adapted to airway epithelial cells (AEC) to sufficiently differentiate apoptotic and necrotic AEC. Specifically, cell lines and primary AEC (n = 12) were permeabilized or infected with rhinovirus 1b (RV1b) over 48 h. Cell death was then measured via annexin V/propidium iodide (A5/PI) or annexin V/TO-PRO-3 (A5/TP3) staining using a novel flow cytometry and gating methodology adapted to AEC. We show that A5/PI staining could not sufficiently differentiate between types of cell death following RV1b infection of primary AEC. However, A5/TP3 staining was able to distinguish six cell death populations (viable, necrotic, debris, A5⁺ apoptotic, A5^- apoptotic, apoptotic bodies) after permeabilization or infection with RV1b, with phenotypic differences were observed in apoptotic populations. Collectively, using a staining and gating strategy never adapted to AEC, A5/TP3 could accurately differentiate and quantify viable, necrotic, and apoptotic AEC following RV1b infection.

There 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.

Cytokinesis 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 (R ² = 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.

Hepatic steatosis is a metabolic disease, characterized by selective and progressive accumulation of lipids in liver, leading to progressive non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and cirrhosis. The existing in vitro models of hepatic steatosis to elucidate the molecular mechanisms behind the onset of hepatic steatosis and to profile small molecule modulators uses lipid loaded primary hepatocytes, and cell lines like HepG2. The limitation of these models includes high variability between the different donor samples, reproducibility, and translatability to physiological context. An in vitro human hepatocyte derived model that mimics the pathophysiological changes seen in hepatic steatosis may provide an alternative tool for pre-clinical drug discovery research. We report the development of an in vitro experimental model of hepatic steatosis using human induced pluripotent stem cell (iPSC) derived hepatocytes like cells (HLC), loaded with lipids. Our data suggests that HLC carry some of the functional characteristics of primary hepatocytes and are amenable for development of an in vitro steatosis model using lipid loading method. The in vitro experimental model of hepatic steatosis was further characterized using biomarker analysis and validated using telmisartan. With some refinement and additional validation, our in vitro steatosis model system may be useful for profiling small molecule inhibitors and studying the mechanism of action of new drugs.

Research on melanogenesis, its regulation in health and disease, and the discovery of new molecules with pigmenting and depigmenting activities use different models. Here we standardize a protocol based on previous ones using primary human melanocytes and keratinocytes in co-cultures, in which melanogenesis was induced under mild conditions by the addition of tyrosine plus ammonium chloride (NH₄Cl). The expression of MITF, TYR, TYRP1, and Melan-A as well as melanin content were measured. Furthermore, we extended this study to a reconstructed 3D model. Pigmentation was visually observable and melanosomes were identified by Fontana-Masson staining by the addition of tyrosine plus NH₄Cl during the stratification phase. The 2D and 3D protocols proposed here circumvent limitations of previous models, using human primary cells and mild conditions for melanogenesis. These protocols offer a viable, robust, simple, and animal-free investigational option for human skin pigmentation studies and screening tests for new compounds that modulate pigmentation.

Cancer immunotherapy is a rapidly advancing and viable approach to treating cancer along with more traditional forms of therapy. Real-time cell analysis technologies that examine the dynamic interactions between cancer cells and the cells of the immune system are becoming more important for assessment of novel therapeutics. In this report, we use the IncuCyte^® imaging system to study the killing potential of various immune cells on cancer cell lines. The IncuCyte^® system tracks living cells, labeled by a red fluorescent protein, and cell death, as indicated by the caspase-3/7 reagent, which generates a green fluorescent signal upon activation of apoptotic pathways. Despite the power of this approach, obtaining commercially fluorescent cancer cell lines is expensive and limited in the range of cell lines that are available. To overcome this barrier, we developed an inexpensive method using a lentiviral construct expressing nuclear localized mKate2 red fluorescent protein to stably label cancer cells. We demonstrate that this method is effective in labeling a wide variety of cell lines, allowing for analyses of different cancers as well as different cell lines of the same type of cancer.