细胞生物学


分类

现刊
0 Q&A 82 Views Jul 20, 2025

Accurate measurement of protein translation rates is crucial for understanding cellular processes and disease mechanisms. However, existing methods for quantifying translation rates in yeast cells are limited. Here, we present a streamlined protocol for measuring protein translation rates in Saccharomyces cerevisiae using the methionine analog L-azidohomoalanine (AHA), which is the L isoform of this synthetic amino acid, and fluorophore-labeled alkyne dye-based Click chemistry. Our method involves incorporating AHA into newly synthesized proteins, followed by detection using confocal microscopy, flow cytometry, and SDS-PAGE. We validated our protocol by measuring translation rates under various stress conditions, including heat stress, endoplasmic reticulum (ER) stress induced by tunicamycin, and translation inhibition by cycloheximide. Confocal microscopy revealed differential AHA incorporation and fluorescence intensity across conditions. Flow cytometry quantitatively confirmed significant increases in translation rates under heat stress and decreases under ER stress compared to unstressed conditions at 6 and 24 h post-treatment. Imaging of gels under fluorescence detectors following SDS-PAGE further visualized newly synthesized proteins, with no detectable translation after cycloheximide treatment. Our protocol offers enhanced precision and selectivity compared to existing methods for mammalian cells and represents the first standardized approach for measuring translation rates in yeast. Despite limitations in required specialized equipment and expertise, this method holds promise for diverse applications in biotechnology and biomedical research, enabling investigations into protein synthesis regulation in yeast systems.

0 Q&A 366 Views Jul 20, 2025

The skin microbiome, a diverse community of microorganisms, plays a crucial role in maintaining skin health and homeostasis. Traditional studies have relied on two-dimensional (2D) models, which fail to recreate the complex three-dimensional (3D) architecture and cellular interactions of in vivo human skin, and animal models, which have species-specific physiology and accompanying ethical concerns. Consequently, both types of models fall short in accurately replicating skin physiology and understanding its complex microbial interactions. Three-dimensional bioprinting, an advanced tissue engineering technology, addresses these limitations by creating custom-designed tissue scaffolds using biomaterial-based bioinks containing living cells. This approach provides a more physiologically relevant 3D structure and microenvironment, allowing the incorporation of microbial communities to better reflect in vivo conditions. Here, we present a protocol for 3D bioprinting an in vitro skin infection model by co-culturing human keratinocytes and dermal fibroblasts in a high-viscosity, fibrin-based bioink to mimic the dermis and epidermis. The bioprinted skin tissue was co-infected with Staphylococcus aureus and Staphylococcus epidermidis to mimic bacterial skin disease. Bacterial survival was assessed through colony-forming unit enumeration. By incorporating bacteria, this protocol offers the potential to serve as a more representative in vivo 3D bioprinted skin infection model, providing a platform to study host–microbe interactions, immune responses, and the development of antimicrobial therapeutics.

0 Q&A 54 Views Jul 20, 2025

The HIV-1 reservoir, consisting of transcriptionally silent integrated HIV-1 proviruses, is a major barrier to a cure, as it persists during effective antiretroviral therapy (ART) and is the source of viral rebound upon treatment interruption. Some of the strategies explored for HIV cure focus on the identification of compounds to either reactivate and eliminate the HIV reservoir (“shock and kill”) or to prevent HIV reservoir reactivation and induce deep proviral latency (“block and lock”). Paramount in developing these HIV-1 cure strategies is determining the effect of the compounds on the size of the inducible HIV-1 reservoir in blood from people living with HIV-1 (PWH). Traditionally, viral outgrowth assays have been the primary method to determine the inducible HIV-1 reservoir in CD4+ T cells from PWH. However, these assays are labor-intensive, time-consuming, and often have low sensitivity. We have recently developed the inducible HIV-1 reservoir reduction assay (HIVRRA), a rapid, cost-effective, and sensitive method to measure the impact of compounds on the inducible replication-competent HIV-1 reservoir in total peripheral blood mononuclear cells (PBMCs) from PWH ex vivo. The HIVRRA simultaneously evaluates the effect of test conditions on the size of the inducible replication-competent HIV-1 reservoir as well as the specificity and toxicity of the test strategy. Using total PBMCs instead of purified CD4+ T cells reduces processing time and resource requirements. This makes the HIVRRA a more practical, scalable tool for evaluating potential HIV-1 cure strategies.

0 Q&A 59 Views Jul 20, 2025

PIEZO1 is a mechanically activated ion channel essential for mechanotransduction and downstream signaling in almost all organ systems. Western blotting is commonly used to study the expression, stability, and post-translational modifications of proteins. However, as a large transmembrane protein, PIEZO1 contains extensive hydrophobic regions and undergoes post-translational modifications that increase its propensity for nonspecific protein–protein interactions. As a result, conventional sample preparation methods seem unsuitable for PIEZO1. For example, heating and sonicating transmembrane proteins exposes hydrophobic regions, leading to aggregation, improper detergent interactions, and loss of solubility, ultimately compromising their detection in western blots. To address these challenges, we developed a western blot protocol optimized for human PIEZO1 by preparing lysates consistently at lower temperatures and incorporating strong reducing and alkylation reagents into the western blot lysis buffer to ensure proper protein solubilization and minimal cross-linking. Using the same antibody, we also developed an immunoprecipitation protocol with optimized detergents to maintain the solubilization of native human PIEZO1, enabling the discovery of a new family of auxiliary subunits.

0 Q&A 61 Views Jul 20, 2025

Brightfield microscopy is an ideal application for studying live cell systems in a minimally invasive manner. This is advantageous in long-term experiments to study dynamic cellular processes such as stress response. Depending on the sample type and preparation, the inherent qualities of brightfield microscopy, being very low contrast, can contribute to technical issues such as focal drift, sequencing lags, and complete failure of software autofocus systems. Here, we describe the use of microbeads as a focus aid for long-term live cell imaging to address these autofocus issues. This protocol is inexpensive to implement, without extensive additional sample preparation, and can be used to capture focused images of transparent cells in a label-free manner. To validate this protocol, a widefield inverted microscope was used with software-based autofocus to image overnight in time-lapse format, demonstrating the use of the beads to prevent focal drift in long-term experiments. This improves autofocus accuracy on relatively inexpensive microscopes without using hardware-based focus aids. To validate this protocol, the KNIME logistics software was used to train a random forest model to perform binary image classification.

0 Q&A 64 Views Jul 20, 2025

Cryo-electron tomography (cryo-ET) is the main technique to image the structure of biological macromolecules inside their cellular environment. The samples for cryo-ET must be thinner than 200 nm, which is not compatible with micron-sized cells. A focused ion beam (FIB), in conjunction with a scanning electron microscope (SEM) to navigate the sample, can be used to ablate material from vitrified cells such that a thin lamella remains. However, the preparation of lamellae with a FIB-SEM is blind to the location of specific cellular structures and biomolecules. Furthermore, the thickness and uniformity of lamella, while crucial for high-quality tomograms, cannot be established accurately with the FIB-SEM. These limitations strongly affect the success rate for cryo-ET on FIB-milled lamellae and thereby the total throughput of the workflow. To mitigate these problems, a coincident light, electron, and ion beam cryo-microscope was developed by retrofitting a fluorescence microscope, cryogenic microcooler, and piezo stage on a FIB-SEM. The fluorescence of molecules of interest can be monitored in real time while milling to ensure the final lamella contains the structure of interest. In addition, reflected light microscopy can be used for thickness and quality control of the lamella. In this protocol, we will describe how the coincident microscope can be used to prepare lamellae from vitrified cells.

0 Q&A 73 Views Jul 20, 2025

Counting protein molecules helps reveal the organization of components within cellular structures and the stoichiometries of protein complexes. Existing protein and peptide quantitation methods vary in their complexity. Here, we report a straightforward workflow to measure the absolute number of HaloTag-labeled myosin 10 (Myo10) molecules in U2OS cells. Myo10 is a motor protein that plays a prominent role in cellular protrusion formation. Various biochemical and biological properties of Myo10 are established, but it is not well-defined how many molecules of Myo10 pack into narrow cellular structures called filopodia. We present a workflow for using SDS-PAGE to calibrate Myo10 signal with a reference protein, segmenting epifluorescence microscopy images to map Myo10 intracellular distribution, and interpreting the results to derive biological and functional insights. Our protocol is simple to employ and not only applicable for Myo10 research but also easily adaptable for other biological systems that use HaloTag.

0 Q&A 49 Views Jul 20, 2025

The persistence of the HIV-1 reservoir remains the ultimate obstacle in achieving a cure. Cure strategies targeting the HIV-1 reservoir are under development, and therefore, finding ways to improve the detection of the reservoir is crucial. Several reservoir detection techniques exist to assess different markers of the HIV-1 reservoir, such as PCR-based assays and protein-based flow cytometric methods. We developed a flow cytometry-fluorescent in situ hybridization (flow-FISH) approach that assesses HIV-1 at the transcriptional level. Using a combination of probes that target either the HIV-1 trans-activation response (TAR) region and 5′ long terminal repeat (LTR) or the Gag sequence, our assay distinguishes between infected cells expressing abortive or elongated HIV-1 RNAs. This assay utilizes the branched-DNA method to amplify the fluorescent signal of the hybridized RNA probes and can be used directly for thawed or cultured cells, with the option to include surface antibody staining. Cellular expression of abortive and/or Gag HIV-1 RNAs is measured by flow cytometry. Our flow-FISH approach gives insight into the transcriptional dynamics of the HIV-1 reservoir and allows for the characterization of latently infected cells.

0 Q&A 59 Views Jul 20, 2025

In vitro systems based on Xenopus egg extracts have elucidated many aspects of spindle assembly. Still, numerous unknowns remain, particularly concerning the variation in spindle morphologies. The X. laevis and X. tropicalis egg extract systems, which recapitulate diverse spindle sizes and architectures, serve as ideal tools to investigate the regulation of spindle morphometrics. However, fully understanding spindle architectural differences is hindered by the spindle's size and high microtubule density. Indeed, classical fluorescence microscopy lacks the resolution to detail the organization of spindle microtubules, and although electron tomography can distinguish individual microtubules, segmenting thousands of microtubules and tracking them across dozens of sections remains an unachieved challenge. Therefore, we set out to apply expansion microscopy to the study of Xenopus egg extract spindles. During this process, we realized that optimizing spindle fixation as well was crucial to preserve microtubule integrity. Here, we present an optimized fixation and expansion microscopy protocol that enables the study of spindle architecture in egg extracts of both X. laevis and X. tropicalis. Our method retains the fluorescence of rhodamine tubulins added to the extracts and allows for both pre- and post-expansion immunofluorescence analysis.

0 Q&A 119 Views Jul 20, 2025

Well-differentiated airway epithelial cultures are commonly used to study airway stem cell lineages, ion and fluid transport, respiratory virus infection and replication, and disease mechanisms in vitro. This culture model involves the isolation and expansion of airway stem cells followed by their differentiation at an air–liquid interface (ALI), a process that has been previously documented in humans and mice. Domestic ferrets (Mustela putorius furo) have gained considerable importance in respiratory disease research due to their notable susceptibility to these conditions and their anatomical similarities to humans. Here, we present a comprehensive description of the isolation and culture of stem/progenitor cells from the ferret airway, along with a protocol for their differentiation at the ALI. Our findings have demonstrated that this ferret culture system not only supports the differentiation of the predominant airway epithelial cell types but also facilitates the generation of rare airway epithelial subpopulations, including pulmonary ionocytes, tuft cells, and pulmonary neuroendocrine cells. Additionally, we provide a detailed procedure for measuring transepithelial ion transport relevant to airway diseases, particularly cystic fibrosis. The ability to isolate and culture ferret airway stem cells, combined with ALI differentiation and functional assessment of transepithelial ion transport, offers a powerful platform for evaluating genetic and pharmacologic interventions related to cystic fibrosis.

往期刊物
0 Q&A 115 Views Jul 5, 2025

Zika virus (ZIKV), an arthropod-borne orthoflavivirus, has emerged as a global health concern due to its ability to cause severe fetal neurological disorders, leading to the congenital Zika syndrome (CZS) in neonates. Vertical transmission during pregnancy can alter neural progenitor cell (NPC) proliferation and differentiation and induce apoptosis, leading to microcephaly and other neurodevelopmental abnormalities. While mammalian models have been used to study the impact of ZIKV on NPC behavior, limitations such as high costs, dedicated time, and ethical constraints have fostered the exploration of alternative systems. The zebrafish embryo constitutes an advantageous in vivo model for studying ZIKV neuropathogenesis. Indeed, ZIKV infection phenocopies several features of the CZS while sharing a conserved neuroanatomical layout and offering genetic plasticity and unique accessibility to the infected brain compared to mammals. Here, we describe a protocol for characterizing ZIKV-induced defects of NPCs in this zebrafish model, relying on whole animal flow cytometry.

0 Q&A 123 Views Jul 5, 2025

In vivo two-photon imaging of the mouse brain is essential for understanding brain function in relation to neural structure; however, its application is limited by the size and mechanical stability of conventional cranial windows. Here, we present the procedure of a large-scale cranial window technique based on the nanosheet incorporated into light-curable resin (NIRE) method. This approach utilizes a biocompatible polyethylene-oxide-coated CYTOP (PEO-CYTOP) nanosheet combined with light-curable resin, allowing the window to conform to the brain’s curved surface. The protocol enables long-term, high-resolution, and multiscale imaging—from subcellular structures to large neuronal populations—in awake mice over several months.

0 Q&A 179 Views Jul 5, 2025

The subcellular localization of RNA plays a critical role in various biological processes, including development and stress response. Proximity labeling eases the detection of localized transcripts and protein enrichment compared to previous techniques that rely on biochemical isolation of subcellular structures. The rapid reaction and small labeling radius of APEX2 make it an attractive alternative to other proximity labeling approaches, such as BioID. However, we found that standard protocols for APEX proximity labeling fail in human induced pluripotent stem cells. Moreover, standard protocols yield heterogeneous labeling of biomolecules across single cells in MCF10A breast epithelial cells. Our results indicate that low biotin permeability in these cell lines is the main cause for failed or inefficient labeling. This protocol outlines improved labeling by combining the rapid hydrogen peroxide-driven APEX2 reaction with the addition of a mild detergent during biotin incubation. This adaptation leads to efficient proximity labeling in hiPSCs and more homogeneous biotinylation across single cells in MCF10As. The adapted protocol extends the use of APEX2 proximity labeling to cell lines with poor biotin permeability.

0 Q&A 278 Views Jul 5, 2025

Since the discovery that astrocytes are characterized by Ca2+-based excitability, investigating the function of these glial cells within the brain requires Ca2+ imaging approaches. The technical evolution from chemical fluorescent Ca2+ probes with low cellular specificity to genetically encoded indicators (GECIs) has enabled detailed analysis of the spatial and temporal features of intracellular Ca2+ signal. Different imaging methodologies allow the extraction of distinct information on calcium signals in astrocytes from brain slices, with resolution ranging from cell populations to single cells up to subcellular domains.

Here, we describe 2-photon laser scanning microscopy (2PLSM) Ca2+ imaging in astrocytes from the somatosensory cortex (SSCx) of adult mice in ex vivo acute cortical slices, performed using two genetically encoded Ca2+ indicators, i.e., cytosolic GCaMP6f and endoplasmic reticulum-targeted G-CEPIA1er. The main advantage of the 2PLSM technique, compared to single-photon microscopy, is the possibility to go deeper in the tissue while avoiding photodamage, by limiting laser excitation to a single focal plane. The fluorescent signal of the indicator is analyzed offline in different compartments—soma, proximal processes, and microdomains—for GCaMP6f experiments and in the perinuclear, somatic area for G-CEPIA1er. The analysis of Ca2+ signal from different compartments, although not providing a value of absolute concentration, allows a critical comparison of the degree of astrocyte activation between different experimental conditions or mouse models. Moreover, the analysis of G-CEPIA1er signal, which reveals metabotropic receptor activation as a dynamic decrease in free Ca2+ in the endoplasmic reticulum (ER), can provide information on possible alterations in this critical second messenger pathway in astrocytes, including, for example, steady-state ER Ca2+ levels and kinetics of Ca2+ release.

0 Q&A 155 Views Jul 5, 2025

Mitochondria are dynamic organelles with essential roles in energetics and metabolism. Several metabolites are common to both the cytosolic and mitochondrial fractions of the cell. The compartmentalization of metabolites within the mitochondria allows specialized uses for mitochondrial metabolism. Inorganic phosphate (Pi) is one such critical metabolite required for ATP synthesis, via glycolysis and mitochondrial oxidative phosphorylation. Estimating total cellular Pi levels cannot distinguish the distribution of Pi pools across different cellular compartments, such as the cytosol and mitochondria, and therefore separate the contributions made toward glycolysis or other cytosolic metabolic processes vs. mitochondrial outputs. Quantifying Pi pools in mitochondria can therefore be very useful toward understanding mitochondrial metabolism and phosphate homeostasis. Here, we describe a protocol for the fairly rapid, efficient isolation of mitochondria from Saccharomyces cerevisiae by immunoprecipitation for quantitative estimation of mitochondrial and cytosolic Pi pools. This method utilizes magnetic beads to capture FLAG-tagged mitochondria (Tom20-FLAG) from homogenized cell lysates. This method provides a valuable tool to investigate changes in mitochondrial phosphate dynamics. Additionally, this protocol can be coupled with LC–MS approaches to quantitatively estimate mitochondrial metabolites and proteins and can be similarly used to assess other metabolite pools that are partitioned between the cytosol and mitochondria.

0 Q&A 132 Views Jul 5, 2025

Adoptive immune cell therapy, especially chimeric antigen receptor T (CAR-T) cells, has emerged as a promising strategy in solid tumor treatment, owing to its unique ability to specifically recognize and effectively eliminate tumor cells. Patient-derived organoids (PDOs) offer a robust and physiologically relevant platform for assessing the safety and efficacy of CAR-T-cell-based therapies. We now describe a detailed protocol for an in vitro evaluation system based on the co-culture of PDOs and CAR-T cells. This system encompasses the establishment of tumor organoids from patient tumor specimens, the isolation of T cells from matched peripheral blood mononuclear cells (PBMCs), and the generation of antigen-specific CAR-T cells. Through the use of fluorescent labeling to visualize different cells and apoptosis-related events post-interaction, along with quantitative analyses of T-cell proliferation, tumor organoid apoptosis, and the secretion of immune effector molecules, this system enables a robust and multifaceted evaluation of CAR-T cell cytotoxicity in vitro. Collectively, this co-culture system provides a systematic and reproducible in vitro platform for evaluating the functional activity of CAR-T cells and advancing research in tumor immunology and immunotherapy.

0 Q&A 101 Views Jul 5, 2025

Over the lifespan of an individual, brain function requires adjustments in response to environmental changes and learning experiences. During early development, neurons overproduce neurite branches, and neuronal pruning removes the unnecessary neurite branches to make a more accurate neural circuit. Drosophila motoneurons prune their intermediate axon bundles rather than the terminal neuromuscular junction (NMJ) by degeneration, which provides a unique advantage for studying axon pruning. The pruning process of motor axon bundles can be directly analyzed by real-time imaging, and this protocol provides a straightforward method for monitoring the developmental process of Drosophila motor neurons using live cell imaging.

0 Q&A 228 Views Jul 5, 2025

The cAMP-dependent protein kinase (PKA) is one of the most extensively distributed kinases among intracellular signal cascades, with a pivotal role in the regulation of various processes, including the capacitation of sperm cells. Traditional assessments of PKA activity rely on the utilization of [γ-32P] ATP and the Kemptide peptide as a substrate. This strategy presents several major drawbacks, including high costs and health risks derived from the manipulation of radioactive isotopes. In this work, we introduce an enhanced non-radioactive assay to quantify PKA activity, termed kinase mobility shift assay (KiMSA), based on the use of a fluorescent-labeled Kemptide (Kemptide-FITC). Once the kinase reaction is terminated, the products can be easily resolved through electrophoresis on an agarose gel and quantified by fluorescence densitometry. We show that KiMSA is suitable for isolated PKA as well as for the enzyme in cell extracts. In addition, it enables quantification of PKA activity during the progression of mouse sperm capacitation. Furthermore, the assay enables monitoring the inhibition of PKA with pharmacological inhibitors in live cells. Therefore, the experimental and optimal assay conditions are set so that KiMSA can be used to assess in vitro as well as in vivo PKA activity in sperm cells. Finally, this method allows for measurement of cAMP concentrations, rendering a versatile technique for the study of cAMP/PKA pathways.

0 Q&A 162 Views Jul 5, 2025

Glomerular diseases characterized by injury to post-mitotic epithelial cells called podocytes are a leading cause of chronic kidney disease. Yet, isolating podocytes from the kidney for transcriptomic, proteomic, and metabolomic studies has been a major technical challenge. Protocols utilizing glomerular sieving and laser capture methods are of limited use because they are not podocyte-specific but instead capture all four glomerular cell types. Here, we present a magnetic-activated cell sorting (MACS) method where podocytes are isolated from digested whole kidneys using antibodies specific to extracellular antigens on podocytes. Using microbeaded secondary antibodies binding to the podocyte-specific primary antibodies allows sorting of the podocytes using a magnet. This podocyte-only cell fraction is a unique source of in vivo–derived cells for molecular and cellular experiments.

0 Q&A 123 Views Jul 5, 2025

The fatal motor neuron (MN) disease amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of the phrenic MNs (phMNs) controlling the activity of the diaphragm, leading to death by respiratory failure. Human experimental models to study phMNs are lacking, hindering the understanding of the mechanisms of phMN degeneration in ALS. Here, we describe a protocol to derive phrenic-like MNs from human induced pluripotent stem cells (hiPSC-phMNs) within 30 days. During spinal cord development, phMNs emerge from specific MN progenitors located in the dorsalmost MN progenitor (pMN) domain at cervical levels, under the control of a ventral-to-dorsal gradient of Sonic hedgehog (SHH) signaling and a rostro-caudal gradient of retinoic acid (RA). The method presented here uses optimized concentrations of RA and the SHH agonist purmorphamine, followed by fluorescence-activated cell sorting (FACS) of the resulting MN progenitor cells (MNPCs) based on a cell-surface protein (IGDCC3) enriched in hiPSC-phMNs. The resulting cultures are highly enriched in MNs expressing typical phMN markers. This protocol enables the generation of hiPSC-phMNs and is highly reproducible using several hiPSC lines, offering a disease-relevant system to study mechanisms of respiratory MN dysfunction. While the protocol has been validated in the context of ALS research, it can be adopted to study human phrenic MNs in other research fields where these neurons are of interest.