Cell Biology


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Protocols in Current Issue
0 Q&A 117 Views Nov 20, 2024

Targeted genome editing of human pluripotent stem cells (hPSCs) is critical for basic and translational research and can be achieved with site-specific endonucleases. Cpf1 (CRISPR from Prevotella and Francisella) is a programmable DNA endonuclease with AT-rich PAM sequences. In this protocol, we describe procedures for using a single vector system to deliver Cpf1 and CRISPR RNA (crRNA) for genome editing in hPSCs. This protocol enables indel formation and homologous recombination–mediated precise editing at multiple loci. With the delivery of Cpf1 and a single U6 promoter-driven guide RNA array composed of an AAVS1-targeting and a MAFB-targeting crRNA array, efficient multiplex genome editing at the AAVS1 (knockin) and MAFB (knockout) loci in hPSCs could be achieved in a single experiment. The edited hPSCs expressed pluripotency markers and could differentiate into neurons in vitro. This system also generated INS reporter hPSCs with a 6 kb cassette knockin at the INS locus. The INS reporter cells can differentiate into β-cells that express tdTomato and luciferase, permitting fluorescence-activated cell sorting of hPSC-β-cells. By targeted screening of potential off-target sequences that are most homologous to crRNA sequences, no off-target mutations were detected in any of the tested sequences. This work provides an efficient and flexible system for precise genome editing in mammalian cells including hPSCs with the benefits of less off-target effects.

0 Q&A 56 Views Nov 20, 2024

The eukaryotic cytoskeleton is formed in part by microtubules, which are relatively rigid filaments with inherent structural polarity. One consequence of this polarity is that the two ends of a microtubule have different properties with important consequences for their cellular roles. These differences are often challenging to probe within the crowded environment of the cell. Fluorescence microscopy–based in vitro assays with purified proteins and stabilized microtubules have been used to characterize polarity-dependent and end-specific behaviors. These assays require ways to visualize the polarity of the microtubules, which has previously been achieved either by the addition of fluorescently tagged motor proteins with known directionality or by fluorescently polarity marking the microtubules themselves. However, classical polarity-marking protocols require a particular chemically modified tubulin and generate microtubules with chemically different plus and minus segments. These chemical differences in the segments may affect the behavior of interacting proteins of interest in an undesirable manner. We present here a new protocol that uses a previously characterized, reversibly binding microtubule plus-end capping protein, a designed ankyrin repeat protein (DARPin), to efficiently produce polarity-marked microtubules with different fluorescently labeled, but otherwise biochemically identical, plus- and minus-end segments.

0 Q&A 57 Views Nov 20, 2024

In plants, the first interaction between the pollen grain and the epidermal cells of the stigma is crucial for successful reproduction. When the pollen is accepted, it germinates, producing a tube that transports the two sperm cells to the ovules for fertilization. Confocal microscopy has been used to characterize the behavior of stigmatic cells post-pollination [1], but it is time-consuming since it requires the development of a range of fluorescent marker lines. Here, we propose a quick, high-resolution imaging protocol using tabletop scanning electron microscopy. This technique does not require prior sample fixation or fluorescent marker lines. It effectively captures pollen grain behavior from early hydration (a few minutes after pollination) to pollen tube growth within the stigma (1 h after pollination) and is particularly efficient for tracking pollen tube paths.

0 Q&A 39 Views Nov 20, 2024

Lysosome-related organelles (LROs) are a class of heterogeneous subcellular organelles conserved in eukaryotes, performing various functions. An important function of LROs is to mediate phosphorus and metal homeostasis. Chlamydomonas reinhardtii serves as a model organism for investigating metal ion metabolism. Considering that LROs contain polyphosphate and various metal elements, the purification strategy is based on their higher density by fractionating cell lysate through OptiPrep density gradient ultracentrifugation. Here, we optimized a method for purifying LROs from C. reinhardtii cells that have reached stationary phase (sta-LROs) or are overloaded with iron (Fe-LROs). Our protocol provides technical support for further investigations on the biogenesis and function of LROs in C. reinhardtii.

0 Q&A 88 Views Nov 20, 2024

Protein carbonylation has been known as the major form of irreversible protein modifications and is also widely used as an indicator of oxidative stress in the biological environment. In the presence of oxidative stress, biological systems tend to produce large amounts of carbonyl moieties; these carbonyl groups do not have particular UV-Vis and fluorescence spectroscopic characteristics that we can differentiate, observe, and detect. Thus, their detection and quantification can only be performed using specific chemical probes. Commercially available fluorescent probes to detect specific carbonylation in biological systems have been used, but their chemical portfolio is still very limited. This protocol outlines the methods and procedures employed to synthesize a probe, (E,Z)-2-(2-(2-hydroxybenzylidene)hydrazonyl)-5-nitrophenol (2Hzin5NP), and assess its impact on carbonylation in human cells. The synthesis involves several steps, including the preparation of its hydrazone compounds mimicking cell carbonyls, 2-Hydrazinyl 5-nitrophenol, (E,Z)-2-(2-ethylidenehydrazonyl)-5-nitrophenol, and the final product (E,Z)-2-(2-(2-hydroxybenzylidene)hydrazonyl)-5-nitrophenol. The evaluation of fluorescence quantum yield and subsequent cell culture experiments are detailed for the investigation of 2Hzin5NP effects on cell proliferation and carbonylation.

0 Q&A 59 Views Nov 20, 2024

Alpha-protein kinase 1 (ALPK1) is normally activated by bacterial ADP-heptose as part of the innate immune response, leading to the initiation of downstream signalling events that culminate in the activation of transcription factors such as NF-κB and AP-1. In contrast, disease-causing mutations in ALPK1 that cause ROSAH syndrome or spiradenoma allow ALPK1 to be activated in cells in the absence of bacterial infection (i.e., without ADP-heptose). This protocol describes a semi-quantitative reporter assay based on ALPK1 knockout HEK-Blue cells that measures the activity of transfected wildtype and disease-causing forms of ALPK1 by virtue of their ability to activate the transcription factors NF-κB and AP-1. These cells express a synthetic gene encoding alkaline phosphatase under the control of an NF-κB/AP-1-dependent promoter, and consequently, the activation of ALPK1 leads to the production of alkaline phosphatase, which is secreted into the culture media and can be measured colorimetrically at 645 nm after the addition of a detection reagent.

0 Q&A 122 Views Nov 20, 2024

This study explores the novel application of pyronin Y for fluorescently labeling extracellular matrices (ECMs) and gelatin cryogels, providing a simple and reliable method for laser scanning confocal microscopy. Pyronin Y exhibited remarkable staining ability of the porous structures of gelatin cryogels, indicating its potential as a reliable tool for evaluating such biomaterials. Confocal imaging of pyronin Y–stained cryogels produced high signal-to-noise ratio images suitable for quantifying pores using Fiji/Image J. Importantly, pyronin Y enabled effective dual-color imaging of cryogel-labeled mesenchymal stem cells, expanding its utility beyond traditional RNA assays. Traditional staining methods like Mason’s trichrome and Sirius Red have limitations in cryogel applications. Pyronin Y emerges as a powerful alternative due to its water solubility, minimal toxicity, and stability. Our results demonstrate pyronin Y’s ability to specifically stain gelatin cryogel's porous structures, surpassing its weak staining of ECMs in 2D. Confocal imaging revealed enduring staining even under rigorous scanning, with no notable photobleaching observed. Furthermore, pyronin Y's combination with Alexa Fluor 647 for dual-color imaging showed promising results, validating its versatility in fluorescence microscopy. In conclusion, this study establishes pyronin Y as a cost-effective and rapid option for fluorescent staining of gelatin cryogels. Its simplicity, efficacy, and compatibility with confocal microscopy make it a valuable tool for characterizing and evaluating gelatin-based biomaterials, contributing significantly to the field of cryogel imaging. The study opens new avenues for dual-color imaging in biomaterial research and tissue engineering, advancing our understanding of cellular interactions within scaffolds.

0 Q&A 429 Views Nov 20, 2024

Primary human intestinal stem cells (ISCs) can be cultured and passaged indefinitely as two-dimensional monolayers grown on soft collagen. Culturing ISCs as monolayers enables easy access to the luminal side for chemical treatments and provides a simpler topology for high-resolution imaging compared to cells cultured as three-dimensional organoids. However, the soft collagen required to support primary ISC growth can pose a challenge for live imaging with an inverted microscope, as the collagen creates a steep meniscus when poured into wells. This may lead to uneven growth toward the center of the well, with cells at the edges often extending beyond the working distance of confocal microscopes. We have engineered a 3D-printed collagen mold that enables the preparation of chamber slides with flat, smooth, and reproducible thin collagen layers. These layers are adequate to support ISC growth while being thin enough to optimize live imaging with an inverted microscope. We present methods for constructing the collagen press, preparing chamber slides with pressed collagen, and plating primary human ISCs for growth and analysis.

0 Q&A 63 Views Nov 20, 2024

ALPK1 is an atypical protein kinase that is activated during bacterial infection by ADP-heptose and phosphorylates TIFA to activate a cell signaling pathway. In contrast, specific mutations in ALPK1 allow it to also be activated by endogenous human nucleotide sugars such as UDP-mannose, leading to the phosphorylation of TIFA in the absence of infection. This protocol describes a quantitative, cell-free phosphorylation assay that can directly measure the catalytic activity of wildtype and disease-causing ALPK1 in the presence of different nucleotide sugars. In this method, overexpressed ALPK1 is first immunoprecipitated from the extracts of ALPK1 knockout HEK-Blue cells transfected with plasmids encoding either FLAG-tagged wildtype or mutant ALPK1, and then subjected to a radioactive phosphorylation assay in which the phosphorylation of purified GST-tagged TIFA by ALPK1 is quantified by measuring the incorporation of radioactivity derived from radiolabeled ATP.

Protocols in Past Issues
0 Q&A 213 Views Nov 5, 2024

Osteoclasts are terminally differentiated multinucleated giant cells that mediate bone resorption and regulate skeletal homeostasis under physiological and pathological states. Excessive osteoclast activity will give rise to enhanced bone resorption, being responsible for a wide range of metabolic skeletal diseases, ranging from osteoporosis and rheumatoid arthritis to tumor-induced osteolysis. Therefore, the construction of in vitro models of osteoclast-mediated bone resorption is helpful to better understand the functional status of osteoclasts under (patho)physiological conditions. Notably, it is essential to provide an in vivo–relevant bone substrate that induces osteoclasts to generate authentic resorption lacunae and excavate bone. Here, we summarize the experimental design of a reproducible and cost-effective method, which is suitable for evaluating the regulatory mechanisms and influence of molecular agonists and antagonists as well as therapeutics on osteoclast-mediated bone-resorbing activity.

0 Q&A 220 Views Nov 5, 2024

The parasympathetic nervous system is essential for salivary gland development and functionality. Parasympathetic neuron (parasymN) innervation is the main neural network that controls salivary secretion. Therefore, an exclusive model to study parasympathetic neurons and salivary gland tissue circuitry will significantly improve the understanding of the role of parasymN activation on salivary regulation. Harvesting primary rodent parasymNs is challenging due to their body-wide disbursed location. Similarly, the salivary glands are distributed in various locations around and within the oral cavity. Here, we present a coculture model system using human pluripotent stem cell (hPSC)-derived parasymNs and primary mouse von Ebner’s gland cells. We previously reported the first protocol to robustly generate human parasymNs from hPSCs through the Schwann cell precursor (SCP) lineage. The hPSC-parasymNs are functional and have been applied to model several autonomic disorders. We also used a Sox10-Cre::tdTomato (hereafter referred to as RFP) reporter mouse line, which labeled von Ebner’s glands, a type of minor salivary gland connected to the trough of circumvallate and foliate taste papillae. This labeling allowed for visualization and efficient isolation of primary tissues in young adult mice (8–10 weeks). By coculturing the two tissues, human parasymNs control mouse salivary gland cell growth and activation. Both parasymNs and primary salivary gland cells can be frozen and stocked at early stages of differentiation and isolation, making applications easier. This novel coculture model system could also be used to model and study related human diseases in the future, such as dry mouth syndrome.

0 Q&A 184 Views Nov 5, 2024

Plants use CO2, water, and light energy to generate carbohydrates through photosynthesis. During daytime, these carbohydrates are polymerized, leading to the accumulation of starch granules in chloroplasts. The catabolites produced by the degradation of these chloroplast starch granules are used for physiological responses and plant growth. Various staining methods, such as iodine staining, have previously been used to visualize the accumulation of chloroplast starch granules; however, these staining methods cannot be used to image live cells and/or provide confocal images with non-specific signals. In this study, we developed a new imaging method for the fluorescent observation of chloroplast starch granules in living plant cells by staining with fluorescein, a widely available fluorescent dye. This simple staining method, which involves soaking a leaf disk in staining solution, shows high specificity in confocal images. Fluorescent images of the stained tissue allow the cellular starch content of living cells to be quantified with the same level of accuracy as a conventional biochemical method (amyloglucosidase/α-amylase method). Fluorescein staining thus not only enables the easy and clear observation of chloroplast starch granules but also allows for precise quantification in living cells.

0 Q&A 237 Views Nov 5, 2024

Drug-induced hearing injury (ototoxicity) is a common, debilitating side effect of many antibiotic regimens that can be worsened by adverse drug interactions. Such adverse drug interactions are often not detected until after drugs are already on the market because of the difficulty of measuring all possible drug combinations. While in vivo mammalian assays to screen for ototoxic damage exist, they are currently time-consuming, costly, and limited in throughput, which limits their utility in assessing drug interaction outcomes. To facilitate more rapid quantification of ototoxicity and assessment of adverse drug interactions that impact ototoxicity, we have developed a high-throughput workflow we call parallelized evaluation of protection and injury for toxicity assessment (PEPITA). PEPITA uses zebrafish larvae to quantify ototoxic damage and protection. Previous work has shown that hair cells (HCs) in the zebrafish lateral line are very similar to human inner ear HCs, meaning zebrafish are a viable model to test drug-induced ototoxicity. In PEPITA, we expose zebrafish larvae to different combinations of drugs, fluorescently label the HCs, and subsequently use microscopy to quantify the brightness of the fluorescently labeled HCs as an assay for ototoxic damage and hair-cell viability. PEPITA is a reproducible, low-cost, technically accessible, and high-throughput assay. These advantages allow many experiments to be conducted in parallel, paving the way for systematic evaluation of drug-induced hearing injury and other multidrug interactions.

0 Q&A 110 Views Nov 5, 2024

This paper presents a refined, user-friendly protocol for using boron-dipyrromethene (BODIPY) to assess and quantify foam cells and lipid droplet–accumulating microglia (LDAM) in mouse brain tissue. The protocol aims to enhance existing methodologies by offering precise and efficient evaluation of foam cells and LDAM burden in various neuropathological conditions linked to lipid metabolism and neuroinflammation. A notable challenge in analyzing tissue from mouse models of these neurodegenerative disorders is the interference caused by the autofluorescent molecule lipofuscin. Our protocol addresses this issue with specific steps that effectively distinguish BODIPY fluorescence from lipofuscin autofluorescence, using advanced imaging techniques and filter settings to ensure accurate and reliable analysis. By providing a straightforward and accessible method, this research aims to facilitate the broader adoption of BODIPY-based techniques for detailed foam cell and LDAM analysis in mouse brain tissue, potentially enhancing diagnostic capabilities and deepening our understanding of how these cells contribute to neurodegenerative disease mechanisms.

0 Q&A 249 Views Oct 20, 2024

Dengue virus (DENV), a common and prevalent mosquito-borne endemic disease, is caused by four serotypes (DENV-1–4) and has spread rapidly on a global scale over the past decade. A crucial step in the development of antiviral therapeutics requires the utilization of in vitro cell-based techniques, such as plaque assays and focus-forming assays (FFA) for virus quantification. Vero cells have been widely used for FFA and plaque assay; however, there are instances when their efficacy and efficiency in the detection of certain clinical DENV isolates are low. Here, we showed that BHK-21 cells are more sensitive than Vero cells in the detection of all DENV-1–4 plaques and foci. In addition, we developed an improved FFA protocol for the quantification of all four DENV serotypes. Using a pan-flavivirus envelope (E) antibody, we reduce the possibility of false positives by defining a focus to consist of a minimum of eight infected cells. We outlined a protocol using the Operetta® high-content imaging system to automate the digital capture of these infected cells. A pipeline was also designed using the CellProfilerTM automated image analysis software to detect these foci. We then compare the results of the improved FFA with plaque assay. Notably, the improved FFA detected clear foci of the DENV-4 strain that does not form distinct plaques. We subsequently demonstrated the potential application of the improved FFA protocol in antiviral testing, utilizing a nucleoside inhibitor of DENV, NITD008 as a control. The protocol is amenable to a diverse array of applications, including high-throughput compound screening (HTS).

0 Q&A 422 Views Oct 20, 2024

The adoptive transfer of autologous, long-lived, gene-repaired T cells is a promising way to treat inherited T-cell immunodeficiencies. However, adoptive T-cell therapies require a large number of T cells to be manipulated and infused back into the patient. This poses a challenge in primary immunodeficiencies that manifest early in childhood and where only small volumes of blood samples may be available. Our protocol describes the ex vivo expansion of potentially long-lived human T memory stem cells (TSCM), starting from a limited number of peripheral blood mononuclear cells (PBMCs). Using the perforin gene as an example, we provide detailed instructions for precise gene repair of human T cells and the expansion of TSCM. The efficiency of precise gene repair can be increased by suppressing unintended non-homologous end-joining (NHEJ) events. Our protocol yields edited T-cell populations that are ready for phenotyping, genome-wide off-target analysis, and functional characterization.

0 Q&A 340 Views Oct 20, 2024

MreB is a prokaryotic actin homolog. It is essential for cell shape in the majority of rod-shaped cell-walled bacteria. Structural and functional characterization of MreB protein is important to understand the mechanism of ATP-dependent filament dynamics and membrane interaction. In vitro studies on MreBs have been limited due to the difficulty in purifying the homogenous monomeric protein. We have purified MreB from the cell-wall-less bacteria Spiroplasma citri, ScMreB5, using heterologous expression in Escherichia coli. This protocol provides a detailed description of purification condition optimization that led us to obtain high concentrations of stable ScMreB5. Additionally, we have provided a protocol for detecting the presence of monovalent ions in the ScMreB5 AMP-PNP-bound crystal structure. This protocol can be used to obtain a high yield of ScMreB5 for carrying out biochemical and reconstitution studies. The strategies used for ScMreB5 show how optimizing buffer components can enhance the yield and stability of purified protein.

0 Q&A 226 Views Oct 20, 2024

The mammary gland undergoes functional, developmental, and structural changes that are essential for lactation and reproductive processes. An overview of such unique tissue can offer clearer insights into mammary development and tumorigenesis. Compared to traditional methods, mouse mammary gland whole mount is a pivotal technique that provides three-dimensional structural perspectives on gland morphology and developmental stages, offering an inexpensive and accessible approach. This protocol outlines the tissue isolation of the mouse mammary gland and provides detailed instructions for whole-mount staining and analysis. Mammary gland tissues are carefully dissected from euthanized mice and stained with Carmine Alum to highlight the ductal structures, enabling detailed visualization of the branching patterns and morphological changes. Light microscopy is used to capture a panoramic image of the stained mammary gland, enabling the quantitative analysis of terminal end buds (TEBs) and bifurcated TEBs to further investigate mammary gland remodeling. This method can provide invaluable insights, particularly in the study of mammary gland morphogenesis and tumorigenesis, underscoring its significance in both basic research and clinical applications.

0 Q&A 262 Views Oct 20, 2024

Endometrial cancer (EC) is the leading cause of gynecologic cancer morbidity and mortality in the U.S. Despite advancements in cancer research, EC death rates are increasing, particularly high-grade endometrial cancers. The development of three-dimensional (3D) patient-derived organoid (PDO) models for EC is crucial, as they provide a more accurate representation of the biological and genetic complexity of a patient’s tumor compared to traditional 2D cell lines. Here, we describe a protocol for cultivating PDO models from normal endometrium and EC across different EC subtypes. These EC PDO models can be expanded across multiple passages and facilitate the exploration of tumor behavior and drug responses, thereby advancing our understanding of the disease and potentially leading to more effective and individualized novel therapeutic strategies.




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