Bio-protocol最新文献

When performing expression analysis either for coding RNA (e.g., mRNA) or non-coding RNA (e.g., miRNA), reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used method. To normalize these data, one or more stable endogenous references must be identified. RefFinder is an online web-based tool using four almost universally used algorithms for assessing candidate endogenous references-delta-Ct, BestKeeper, geNorm, and Normfinder. However, the online interface is presently cumbersome and time consuming. We developed an R package, RefSeeker, which performs easy and straightforward RefFinder analysis by enabling raw data import and calculation of stability from each of the algorithms and provides data output tools to create graphs and tables. This protocol uses RefSeeker R package for fast and simple RefFinder stability analysis. Key features Perform stability analysis using five algorithms: Normfinder, geNorm, delta-Ct, BestKeeper, and RefFinder. Identification of endogenous references for normalization of RT-qPCR data. Create publication-ready graphs and tables output. Step-by-step guide dialog window for novice R users.

Platelets play an important role in hemostasis by forming clots and stopping bleeding. In immune thrombotic conditions, platelets and leukocytes are aberrantly activated by pathogenic antibodies resulting in platelet aggregates and NETosis, leading to thrombosis and thrombocytopenia. A simple assay that assesses platelet function and antibody activity is light transmission aggregometry. This assay can be used to determine antibody activity in patients with disorders such as heparin-induced thrombocytopenia (HIT) and vaccine-induced thrombotic thrombocytopenia (VITT). Briefly, for detection of pathogenic antibody, platelet-rich plasma (PRP) is treated with a specific agent (e.g., patient sera or purified patient antibodies) with constant stirring. Upon activation, platelets undergo a shape change and adhere to each other forming aggregates. This causes a reduction in opacity allowing more light to pass through PRP. Light transmission through the cuvette is proportional to the degree of platelet aggregation and is measured by the photocell over time. The advantage of this protocol is that it is a simple, reliable assay that can be applied to assess antibody activity in thrombotic conditions. Light transmission aggregometry does not require the use of radioactive reagents and is technically less demanding compared with ¹⁴C-serotonin release assay, another common assay for detecting antibody activity. Key features • This protocol can be used to assess platelet function and to detect platelet activating antibodies in diseases such as HIT and VITT. • Does not require radioactive reagents, requires an aggregometer; based on the light transmission aggregometry protocol, adapted for detection of VITT and other platelet-activating antibodies. • Two positive controls are required for reliable detection of antibodies in diseases such as HIT/VITT, namely a weak HIT/VITT antibody and a physiological agonist. • For detection of HIT/VITT antibodies, it is essential to use donors known to have platelets reactive to these antibodies to avoid false negative results.

An emerging body of behavioural studies indicates that regular swimming in cold water has positive effects on mental health and wellbeing, such as reducing fatigue, improving mood, and lessening depressive symptoms. Moreover, some studies reported immediate effects of cold-water immersion (CWI) on elevating mood and increasing a positive emotional state. However, the neural mechanisms underlying these effects remain largely unknown. The lack of studies using neuroimaging techniques to investigate how a whole-body CWI affects neural processes has partly resulted from the lack of a tested experimental protocol. Previous protocols administered tonic limb cooling (1-10 °C) while recording functional magnetic resonance (fMRI) signals. However, using very low water temperature constitutes points of contrast to painful experiences that are different from what we experience after a whole-body head-out CWI. In our protocol, healthy adults unhabituated to cold water were scanned twice: immediately before (pre-CWI) and after (post-CWI) immersion in cold water (water temperature 20 °C) for 5 min. We recorded cardiac and ventilatory responses to CWI and assessed self-reported changes in positive and negative affects. Our protocol showed reliable changes in brain connectivity after a short exposure to cold water, thus enabling its use as a tested experimental framework in future studies.

Expansion microscopy is an innovative method that enables super-resolution imaging of biological materials using a simple confocal microscope. The principle of this method relies on the physical isotropic expansion of a biological specimen cross-linked to a swellable polymer, stained with antibodies, and imaged. Since its first development, several improved versions of expansion microscopy and adaptations for different types of samples have been produced. Here, we show the application of ultrastructure expansion microscopy (U-ExM) to investigate the 3D organization of the green algae Chlamydomonas reinhardtii cellular ultrastructure, with a particular emphasis on the different types of sample fixation that can be used, as well as compatible staining procedures including membranes. Graphical overview.

In the field of molecular genetics, DNA extraction protocols and kits are sample-specific and proprietary, preventing lateral distribution among similar facilities from different sectors to alleviate supply shortages during a crisis. Expanding upon previous fast extraction protocols such as alkaline- and detergent-based ones, the use of boiling-hot water to rupture cells, virions, and nuclei, as proposed during the COVID-19 pandemic, might alleviate shortages and costs. Different soft, relatively abundant (highly enriched), and uncomplicated (genomically homogenous and with few inhibitors) biosamples are collected in 1.5 mL tubes, mixed with boiling-hot water, and stirred vigorously, so as to have membranes lysed and proteins deactivated; mechanical disruption may be used as well if necessary. Incubation in boiling water bath for 20-30 min follows. Depending on sample type and quantity, which affects the total extraction volume, 2-5 μL are pipetted off for direct PCR and the same volume for two decimal serial dilutions. The latter are intended to optimize the crude extract to a workable DNA/inhibitor concentration balance for direct PCR. Uncomplicated, highly enriched samples such as mycelial growth in fruits and human swab samples can be processed, contrary to complicated samples such as blood and physically unyielding samples such as plant tissue. The extract can be used for immediate PCR in both benchtop and portable thermocyclers, thus allowing nucleic acid amplification tests (NAAT) being performed in resource-limited settings with low cost and waste footprint or during prolonged crises, where supply chain failures may occur. Key features DNA extraction from different sample types using only boiling water and occasional mechanical assistance. Crude extract serially diluted twice, 10- and 100-fold, to bypass purification and quantification steps. Direct PCR for 2-10 μL of crude lysate and dilutions (conditional to sample type and quantity) to enhance probability of workable DNA-inhibitors' concentrations. Lowers the cost and curtails the overall footprint of testing to increase sustainability in field operations and in standard lab environments under supply chain derailment.

Store-operated Ca²⁺ entry (SOCE) is a ubiquitous Ca2+ signaling modality mediated by Orai Ca²⁺ channels at the plasma membrane (PM) and the endoplasmic reticulum (ER) Ca²⁺ sensors STIM1/2. At steady state, Orai1 constitutively cycles between an intracellular compartment and the PM. Orai1 PM residency is modulated by its endocytosis and exocytosis rates. Therefore, Orai1 trafficking represents an important regulatory mechanism to define the levels of Ca²⁺ influx. Here, we present a protocol using the dually tagged YFP-HA-Orai1 with a cytosolic YFP and extracellular hemagglutinin (HA) tag to quantify Orai1 cycling rates. For measuring Orai1 endocytosis, cells expressing YFP-HA-Orai1 are incubated with mouse anti-HA antibody for various periods of time before being fixed and stained for surface Orai1 with Cy5-labeled anti-mouse IgG. The cells are fixed again, permeabilized, and stained with Cy3-labeled anti-mouse IgG to reveal anti-HA that has been internalized. To quantify Orai1 exocytosis rate, cells are incubated with anti-HA antibody for various incubation periods before being fixed, permeabilized, and then stained with Cy5-labeled anti-mouse IgG. The Cy5/YFP ratio is plotted over time and fitted with a mono-exponential growth curve to determine exocytosis rate. Although the described assays were developed to measure Orai1 trafficking, they are readily adaptable to other PM channels. Key features Detailed protocols to quantify endocytosis and exocytosis rates of Orai1 at the plasma membrane that can be used in various cell lines. The endocytosis and exocytosis assays are readily adaptable to study the trafficking of other plasma membrane channels.

The flux in photosynthesis can be studied by performing ¹³CO₂ pulse labelling and analysing the temporal labelling kinetics of metabolic intermediates using gas or liquid chromatography linked to mass spectrometry. Metabolic flux analysis (MFA) is the primary approach for analysing metabolic network function and quantifying intracellular metabolic fluxes. Different MFA approaches differ based on the metabolic state (steady vs. non-steady state) and the use of stable isotope tracers. The main methodology used to investigate metabolic systems is metabolite steady state associated with stable isotope labelling experiments. Specifically, in biological systems like photoautotrophic organisms, isotopic non-stationary 1¹³C metabolic flux analysis at metabolic steady state with transient isotopic labelling (¹³C-INST-MFA) is required. The common requirement for metabolic steady state, alongside its very short half-timed reactions, complicates robust MFA of photosynthetic metabolism. While custom gas chambers design has addressed these challenges in various model plants, no similar tools were developed for liquid photosynthetic cultures (e.g., algae, cyanobacteria), where diffusion and equilibration of inorganic carbon species in the medium entails a new dimension of complexity. Recently, a novel tailor-made microfluidics labelling system has been introduced, supplying short ¹³CO₂ pulses at steady state, and resolving fluxes across most photosynthetic metabolic pathways in algae. The system involves injecting algal cultures and medium containing pre-equilibrated inorganic ¹³C into a microfluidic mixer, followed by rapid metabolic quenching, enabling precise seconds-level label pulses. This was complemented by a ¹³CO₂-bubbling-based open labelling system (photobioreactor), allowing long pulses (minutes-hours) required for investigating fluxes into central C metabolism and major products. This combined labelling procedure provides a comprehensive fluxome cover for most algal photosynthetic and central C metabolism pathways, thus allowing comparative flux analyses across algae and plants.

Congenital heart disease (CHD) is often associated with myogenic defects. During heart development, cardiomyocyte growth requires essential cues from extrinsic factors such as insulin-like growth factor 2 (IGF-2). To determine whether and how growth factors account for embryonic cardiomyocyte proliferation, isolation followed by culturing of embryonic cardiomyocytes can be utilized as a useful tool for heart developmental studies. Current protocols for isolating cardiomyocytes from the heart do not include a cardiomyocyte-specific reporter to distinguish cardiomyocytes from other cell types. To optimize visualization of cardiomyocyte proliferation, our protocol utilizes a Tnnt2-promoter-driven H2B-GFP knock-in mouse model (TNNT2^H2B-GFP/+) for in vitro visualization of nuclear-tagged cardiomyocyte-specific fluorescence. A cardiomyocyte-specific genetic reporter paired with an effective proliferation assay improves the reproducibility of mechanistic studies by increasing the accuracy of cell identification, proliferated cell counting, and cardiomyocyte tracking. Key features • This protocol refines previous methods of cardiomyocyte isolation to specifically target embryonic cardiomyocytes. • UsesH2B-GFP/+cardiomyocyte reporters as identified by Yan et al. (2016). • Traces cell proliferation with Phospho-Histone 3 (p-H3) assay. • Has applications in assessing the role of growth factors in cardiomyocyte proliferation.

In this article, we provide a method to isolate embryonic melanoblasts from reporter mouse strains. The mice from which these cells are isolated are bred into the ROSA26^mT/mG reporter background, which results in green fluorescent protein (GFP) expression in the targeted melanoblast population. These cells are isolated and purified by fluorescence-activated cell sorting using GFP fluorescence. We also provide a method to culture the purified melanoblasts for further analysis. This method yields > 99% purity melanoblasts specifically targeted, and can be used for a variety of studies, including gene expression, clonogenic experiments, and biological assays, such as viability, capacity for directional migration, or differentiation into melanin-producing melanocytic cells.

Drug biotransformation by the host microbiome can impact the therapeutic success of treatment. In the context of cancer, drug degradation can take place within the microenvironment of the targeted tumor by intratumor bacteria. In pancreatic cancer, increased chemo-resistance against the frontline chemotherapy gemcitabine is thought to arise from drug degradation by the tumor microbiome. This bacterial-drug interaction highlights the need for developing rapid assays for monitoring bacterial gemcitabine breakdown. While chemical approaches such as high-performance liquid chromatography are suitable for this task, they require specialized equipment and expertise and are limited in throughput. Functional cell-based assays represent an alternate approach for performing this task. We developed a functional assay to monitor the rate of bacterial gemcitabine breakdown using a highly sensitive bacterial reporter strain. Our method relies on standard laboratory equipment and can be implemented at high throughput to monitor drug breakdown by hundreds of strains simultaneously. This functional assay can be readily adapted to monitor degradation of other drugs. Key features Quantification of gemcitabine breakdown by incubating bacteria that degrades the drug and subsequently testing the growth of a reporter strain on filtered supernatant. Use of an optimized reporter strain that was genetically engineered to be a non-degrader strain and highly sensitive to gemcitabine. A high-throughput assay performed in microplates that can be adjusted for identifying bacteria with a fast or slow gemcitabine degradation rate. The assay results can be compared to results from a standard curve with known drug concentrations to quantify degradation rate.