Bio-protocol最新文献

Leishmaniasis, a neglected tropical disease, is caused by the intracellular protozoan parasite Leishmania. Upon its transmission through a sandfly bite, Leishmania binds and enters host phagocytic cells, ultimately resulting in a cutaneous or visceral form of the disease. The limited therapeutics available for leishmaniasis, in combination with this parasite's techniques to evade the host immune system, call for exploring various methods to target this infection. To this end, our laboratory has been characterizing how Leishmania is internalized by phagocytic cells through the activation of multiple host cell signaling pathways. This protocol, which we use routinely for our experiments, delineates how to infect mammalian macrophages with either promastigote or amastigote forms of the Leishmania parasite. Subsequently, the number of intracellular parasites, external parasites, and macrophages can be quantified using immunofluorescence microscopy and semi-automated analysis protocols. Studying the pathways that underlie Leishmania uptake by phagocytes will not only improve our understanding of these host-pathogen interactions but may also provide a foundation for discovering additional treatments for leishmaniasis. Key features • This protocol visualizes and quantifies multiple intracellular forms of Leishmania. • It offers flexibility at various points for researchers to introduce modifications according to their study needs.

The roots of herbaceous and woody plants growing in soil are complex structures that are affected by both natural and artificial fungal colonization to various extents. To obtain comprehensive information about the overall distribution of fungi or oomycetes inside a plant root system, rapid, effective, and reliable screening methods are required. To observe both fine roots, i.e., a common site for penetration of fungi and oomycetes, and mature roots, different techniques are required to overcome visual barriers, such as root browning or tissue thickening. In our protocol, we propose using fast, cost-effective, and non-harmful methods to localize fungal or oomycete structures inside plant roots. Root staining with a fluorescent dye provides a quick initial indication of the presence of fungal structures on the root surfaces. The protocol is followed by clearing and staining steps, resulting in a deeper insight into the root tissue positioning, abundance, and characteristic morphological/reproductive features of fungal or oomycete organisms. If required, the stained samples can be prepared by using freeze-drying for further observations, including advanced microscopic techniques. Key features • The protocol enhances tissue-clearing techniques employing KOH or NaOH and is applicable to a broad range of roots from different plant species. • Hydroxides are mixed with hydrogen peroxide to obtain an efficient bleaching solution, which effectively clears roots without causing significant tissue damage. • The protocol could also be used for staining of fungi or oomycetes localized both on the root surface or inside the root tissues. • Simple combination of non-fluorescent methyl blue and fluorescent solophenyl flavine dyes allows the observation of fungal organisms in both brightfield and fluorescence microscopy.

Cells need to migrate along gradients of chemicals (chemotaxis) in the course of development, wound healing, or immune responses. Neutrophils are prototypical migratory cells that are rapidly recruited to injured or infected tissues from the bloodstream. Their chemotaxis to these inflammatory sites involves changes in cytoskeletal dynamics in response to gradients of chemicals produced therein. Neutrophil chemotaxis has been largely studied in vitro; few assays have been developed to monitor gradient responses in complex living tissues. Here, we describe a laser-wound assay to generate focal injury in zebrafish larvae and monitor changes in behaviour and cytoskeletal dynamics. The first step is to cross adult fish and collect and rear embryos expressing a relevant fluorescent reporter (for example, Lifeact-mRuby, which labels dynamic actin) to an early larval stage. Subsequently, larvae are mounted and prepared for live imaging and wounding under a two-photon microscope. Finally, the resulting data are processed and used for cell segmentation and quantification of actin dynamics. Altogether, this assay allows the visualisation of cellular dynamics in response to acute injury at high resolution and can be combined with other manipulations, such as genetic or chemical perturbations. Key features • This protocol is designed to trigger laser wound in zebrafish larvae using two-photon intravital microscopy. • The ability to wound while imaging makes it possible to monitor the behaviour and actin changes of the cells immediately after gradient exposure. • The protocol requires a two-photon microscope for best results. Compared with one-photon laser wounding, the injury is more precise and has better tissue penetration. • The focal nature of the wounds is suitable for studies of neutrophil swarming/aggregation and can be further adapted to infectious settings.

Single-cell RNA sequencing (scRNA-seq) stands as a cutting-edge technology widely used in biological and biomedical research. Existing scRNA-seq methods rely on reverse transcription (RT) and second-strand synthesis (SSS) to convert RNA to cDNA before amplification. However, these methods often suffer from limited RT/SSS efficiency, which compromises the sensitivity of RNA detection. Here, we develop a new method, linearly amplified single-stranded RNA-derived transcriptome sequencing (LAST-seq), which directly amplifies the original single-stranded RNA without prior RT and SSS and offers high-sensitivity RNA detection and a low level of technical noise in single-cell transcriptome analysis. LAST-seq has been applied to quantify transcriptional bursting kinetics in human cells, advancing our understanding of chromatin organization's role in regulating gene expression. Key features • An RNase H/DNA polymerase-based strategy to attach the T7 promoter to single-stranded RNA. • T7 promoter mediated IVT on single stranded RNA template at single cell level.

Neutrophils, constituting 50%-70% of circulating leukocytes, play crucial roles in host defense and exhibit anti-tumorigenic properties. An elevated peripheral blood neutrophil-to-lymphocyte ratio is associated with decreased survival rates in cancer patients. In response to exposure to various antigens, neutrophils release neutrophil granular proteins, which combine to form web-like structures known as neutrophil extracellular traps (NETs). Previously, the relative percentage of NETs was found to be increased in resected tumor tissue samples from patients with gastrointestinal malignancies. The presence of NETs in peripheral blood is indicative of underlying pathological conditions. Hence, employing a non-invasive method to detect NETs in peripheral blood, along with other diagnostic tests, shows potential as a valuable tool not just for identifying different inflammatory disorders but also for assessing disease severity and determining patient suitability for surgical resection. While reliable methods exist for identifying NETs in tissue, accurately quantifying them in whole blood remains challenging. Many previous methods are time-consuming and rely on a limited set of markers that are inadequate for fully characterizing NETs. Therefore, we established a unique sensitive smear immunofluorescence assay based on blood smears to identify NETs in only as little as 2 μL of whole blood. To identify the NET complexes that have enhanced specificities, this combines the use of various antibodies against neutrophil-specific CD15, NET-specific myeloperoxidase (MPO), citrullinated histone H3 (Cit H3), and nuclear DNA. This protocol offers an easy, affordable, rapid, and non-invasive method for identifying NETs; thus, it can be utilized as a diagnostic marker and targeted through various therapeutic approaches for treating human malignancies. Key features • Characterization of neutrophil extracellular traps in whole blood smears through immunofluorescence staining. • Affordable and quantitative approach to neutrophil extracellular trap detection.

Many studies on mosquito biology rely on laboratory-reared colonies, emphasizing the need for standardized protocols to investigate critical aspects such as disease biology, mosquito behavior, and vector control methods. While much knowledge is derived from anthropophilic species from genera like Anopheles, Aedes, and Culex, there is a growing interest in studying mosquitoes that feed on non-human hosts. This interest stems from the desire to gain a deeper understanding of the evolution of diverse host range use and host specificity. However, there is currently a limited number of comprehensive protocols for studying such species. Considering this gap, we present a protocol for rearing Uranotaenia lowii, a mosquito species specialized in feeding on anuran amphibians by eavesdropping on host-emitted sound cues. Additionally, we provide instructions for successfully shipping live specimens to promote research on this species and similar ones. This protocol helps fill the current gap in comprehensive guidelines for rearing and maintaining colonies of anuran host-biting mosquitoes. It serves as a valuable resource for researchers seeking to establish colonies of mosquito species from the Uranotaeniini tribe. Ultimately, this protocol may facilitate research on the evolutionary ecology of Culicidae, as this family has recently been proposed to have originated from a frog-feeding ancestor. Key features • Rearing and maintenance of colonies of non-human host-biting mosquitoes that feed on frogs using host-emitted acoustic cues. • Provides shipping guidelines aimed to enhance the establishment of colonies by new research groups and specimen exchanges between labs.

The eye is a complex organ composed of multiple tissues in anterior and posterior eye segments. Malfunctions of any of these tissues can lead to ocular diseases and loss of vision. A detailed understanding of the ocular anatomy and physiology in animal models and humans contributes to the development of ocular drugs by enabling studies on drug delivery and clearance routes, pharmacokinetics, and toxicity. This protocol provides step-by-step instructions for the extraction and homogenization of ocular tissues for enzymatic and proteomics analyses. Key features • Suitable protocol for the extraction and isolation of ocular tissue from humans and laboratory animals (rabbit, pig, rat, mouse) while minimizing cross-contamination. • Hard or soft tissue homogenates can be prepared efficiently using a Bead Ruptor homogenizer. • Allows to determine the protein contents in prepared homogenates.