细胞生物学


分类

现刊
0 Q&A 55 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.

0 Q&A 80 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 84 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 43 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 35 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 49 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 73 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 61 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 62 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 46 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.

往期刊物
0 Q&A 706 Views Jun 20, 2025

Epithelial tissues form barriers to the flow of ions, nutrients, waste products, bacteria, and viruses. The conventional electrophysiology measurement of transepithelial resistance (TEER/TER) can quantify epithelial barrier integrity, but does not capture all the electrical behavior of the tissue or provide insight into membrane-specific properties. Electrochemical impedance spectroscopy, in addition to measurement of TER, enables measurement of transepithelial capacitance (TEC) and a ratio of electrical time constants for the tissue, which we term the membrane ratio. This protocol describes how to perform galvanostatic electrochemical impedance spectroscopy on epithelia using commercially available cell culture inserts and chambers, detailing the apparatus, electrical signal, fitting technique, and error quantification. The measurement can be performed in under 1 min on commercially available cell culture inserts and electrophysiology chambers using instrumentation capable of galvanostatic sinusoidal signal processing (4 μA amplitude, 2 Hz to 50 kHz). All fits to the model have less than 10 Ω mean absolute error, revealing repeatable values distinct for each cell type. On representative retinal pigment (n = 3) and bronchiolar epithelial samples (n = 4), TER measurements were 500–667 Ω·cm2 and 955–1,034 Ω·cm2 (within the expected range), TEC measurements were 3.65–4.10 μF/cm2 and 1.07–1.10 μF/cm2, and membrane ratio measurements were 18–22 and 1.9–2.2, respectively.

0 Q&A 135 Views Jun 20, 2025

Primary oligodendrocyte cultures are a crucial driving force for in vitro research on oligodendrocytes (OLs) and myelin. Various methods are available to obtain oligodendrocyte lineage cells, primarily from neonatal rodent brains or human induced pluripotent stem cells (iPSCs). In this protocol, we describe a step-by-step procedure for detaching and cryopreserving primary rat oligodendrocyte progenitor cells (OPCs), followed by the thawing, proliferation, and differentiation of the cryopreserved OPCs. After freezing in a serum-free cryopreservation medium, the OPCs can be preserved at -80 °C for up to two months without notable changes in viability, proliferation, or differentiation into mature OLs. Cryopreserved OPCs can be differentiated into mature OLs with robust myelin processes and the capacity to wrap around neuron-mimicking structures. Combined with the author’s method for primary OL culture, which allows for bulk production of OPCs, OPC cryopreservation may substantially improve the efficiency of in vitro OL research.

0 Q&A 155 Views Jun 20, 2025

Osteoarthritis (OA) is the primary cause of joint impairment, particularly in the knee. The prevalence of OA has significantly increased, with knee OA being a major contributor whose pathogenesis remains unknown. Articular cartilage and the synovium play critical roles in OA, but extracting high-quality RNA from these tissues is challenging because of the high extracellular matrix content and low cellularity. This study aimed to identify the most suitable RNA isolation method for obtaining high-quality RNA from microquantities of guinea pig cartilage and synovial tissues, a relevant model for idiopathic OA. We compared the traditional TRIzol® method with modifications to spin column–based methods (TRIspin-TRIzol®/RNeasyTM, RNeasyTM kit, RNAqueousTM kit, and Quick-RNATM Miniprep Plus kit), and an optimized RNA isolation protocol was developed to increase RNA yield and purity. The procedure involved meticulous sample collection, specialized tissue processing, and measures to minimize RNA degradation. RNA quality was assessed via spectrophotometry and RT–qPCR. The results demonstrated that among the tested methods, the Quick-RNATM Miniprep Plus kit with proteinase K treatment yielded the highest RNA purity, with A260:280 ratios ranging from 1.9 to 2.0 and A260:230 ratios between 1.6 and 2.0, indicating minimal to no salt contamination and RNA concentrations up to 240 ng/μL from ⁓20 mg of tissue. The preparation, storage, homogenization process, and choice of RNA isolation method are all critical factors in obtaining high-purity RNA from guinea pig cartilage and synovial tissues. Our developed protocol significantly enhances RNA quality and purity from micro-quantities of tissue, making it particularly effective for RTqPCR in resource-limited settings. Further refinements can potentially increase RNA yield and purity, but this protocol facilitates accurate gene expression analyses, contributing to a better understanding of OA pathogenesis and the development of therapeutic strategies.

0 Q&A 153 Views Jun 20, 2025

Endometritis is a prevalent gynecological condition, often resulting from bacterial infections, which poses significant risks to women’s reproductive health, including recurrent pregnancy loss, spontaneous abortion, and intrauterine adhesions. While conventional in vitro models have provided valuable insights into the pathogenesis of bacterial-induced endometritis, they often fail to replicate the complex cellular architecture and microenvironment of the endometrium due to species-specific differences and variations in the menstrual cycle. In this study, we present a novel organoid-based culture system that establishes a bacterial-induced endometritis model using endometrial organoids derived from primary epithelial cells. This protocol involves culturing endometrial organoids in a Matrigel-based three-dimensional matrix, followed by infection with Escherichia coli at a defined multiplicity of infection (MOI). The model effectively recapitulates key pathological features of bacterial-induced endometritis, including disruption of the epithelial barrier, release of inflammatory cytokines, and cellular damage. By preserving epithelial polarity, this approach offers enhanced physiological relevance, improves host–pathogen interaction studies, and provides a robust platform for evaluating potential therapeutic interventions.

0 Q&A 321 Views Jun 20, 2025

Single-cell RNA sequencing has revolutionized molecular cell biology by enabling the identification of unique transcription profiles and cell transcription states within the same tissue. However, tissue dissociation presents a challenge for non-model organisms, as commercial kits are often incompatible, and current protocols rely on tissue enzymatic digestion for extended periods. Tissue digestion can alter cell transcription in response to temperature and the stress caused by enzymatic treatment. Here, we propose a protocol to stabilize RNA using a deep eutectic solvent (Vivophix, Rapid Labs) prior to tissue dissociation, thereby avoiding transcription changes induced by the process and preventing RNase activity during incubation. We validated this methodology for three medically important insect vectors: Anopheles gambiae, Aedes aegypti, and Lutzomyia longipalpis. Single-cell RNA sequencing using our insect midgut dissociation protocol yielded high-quality sequencing results, with a high number of cells recovered, a low percentage of mitochondrial reads, and a low percentage of ambient RNA—two hallmark standards of cell quality.

0 Q&A 181 Views Jun 20, 2025

The neuromuscular junction (NMJ) is critical for muscle function, and its dysfunction underlies conditions such as sarcopenia and motor neuron diseases. Current protocols for assessing NMJ function often lack standardized stimulation parameters, limiting reproducibility. This study presents an optimized ex vivo method to evaluate skeletal muscle and NMJ function using the Aurora Scientific system, incorporating validated stimulation protocols for both nerve and muscle to ensure consistency. Key steps include tissue preparation in a low-calcium, high-magnesium solution to preserve NMJ integrity, determination of optimal muscle length, and sequential stimulation protocols to quantify neurotransmission failure and intratetanic fatigue. By integrating rigorous standardization, this approach enhances reproducibility and precision, providing a robust framework for investigating NMJ pathophysiology in aging and disease models.

0 Q&A 161 Views Jun 20, 2025

Human brain development relies on a finely tuned balance between the proliferation and differentiation of neural progenitor cells, followed by the migration, differentiation, and connectivity of post-mitotic neurons with region-specific identities. These processes are orchestrated by gradients of morphogens, such as FGF8. Disruption of this developmental balance can lead to brain malformations, which underlie a range of complex neurodevelopmental disorders, including epilepsy, autism, and intellectual disabilities. Studying the early stages of human brain development, whether under normal or pathological conditions, remains challenging due to ethical and technical limitations inherent to working with human fetal tissue. Recently, human brain organoids have emerged as a powerful in vitro alternative, allowing researchers to model key aspects of early brain development while circumventing many of these constraints. Unlike traditional 2D cultures, where neural progenitors and neurons are grown on flat surfaces, 3D organoids form floating self-organizing aggregates that better replicate the cellular diversity and tissue architecture of the developing brain. However, 3D organoid protocols often suffer from significant variability between batches and individual organoids. Furthermore, few existing protocols directly manipulate key morphogen signaling pathways or provide detailed analyses of the resulting effects on regional brain patterning.


To address these limitations, we developed a hybrid 2D/3D approach for the rapid and efficient induction of telencephalic organoids that recapitulate major steps of anterior brain development. Starting from human induced pluripotent stem cells (hiPSCs), our protocol begins with 2D neural induction using small-molecule inhibitors to achieve fast and homogenous production of neural progenitors (NPs). After dissociation, NPs are reaggregated in Matrigel droplets and cultured in spinning mini-bioreactors, where they self-organize into neural rosettes and neuroepithelial structures, surrounded by differentiating neurons. Activation of the FGF signaling pathway through the controlled addition of FGF8 to the culture medium will modulate regional identity within developing organoids, leading to the formation of distinct co-developing domains within a single organoid. Our protocol combines the speed and reproducibility of 2D induction with the structural and cellular complexity of 3D telencephalic organoids. The ability to manipulate signaling pathways provides an additional opportunity to further increase system complexity, enabling the simultaneous development of multiple distinct brain regions within a single organoid. This versatile system facilitates the study of key cellular and molecular mechanisms driving early human brain development across both telencephalic and non-telencephalic areas.

0 Q&A 195 Views Jun 20, 2025

The study of choroidal endothelial cells is essential for understanding the pathological mechanisms underlying choroidal neovascularization and other vision-threatening disorders. Traditional methods for isolating and culturing primary endothelial cells often yield mixed populations or require specialized equipment, limiting their widespread use. Here, we present a straightforward protocol for isolating and culturing primary mouse choroidal endothelial cells. This protocol involves enzymatic digestion of choroidal tissue, magnetic-activated cell sorting (MACS) to enrich CD31+ endothelial cells, and optimized culture conditions to promote cell proliferation and maintain endothelial phenotype. The protocol is strategic, reproducible, and requires minimal specialized equipment, making it accessible for researchers across various fields. By providing a robust method for obtaining pure choroidal endothelial cell cultures, this protocol facilitates the study of cell-specific behaviors and responses, advancing research into choroidal vascular diseases.

0 Q&A 199 Views Jun 20, 2025

The target of rapamycin complex 1 (TORC1) is a highly conserved protein complex whose primary function is to link nutrient availability to cell growth in eukaryotes, particularly nitrogen sources. It was originally identified during the screening of Saccharomyces cerevisiae strains resistant to rapamycin treatment. For its part, S. cerevisiae is well known for being a key model organism in biological research and an essential microorganism for the fermentation of food and beverages. This yeast is widely distributed in nature, with domesticated and wild strains existing. However, little is known about what effects domestication has had on its different phenotypes; for example, how nitrogen sources are sensed for TORC1 activation and what impact domestication has had on TORC1 activation are questions that still have no complete answer. To study the genetic basis of TORC1 activation associated with domestication through approaches such as quantitative trait loci (QTL) mapping or genome-wide association studies (GWAS), and more generally for any study requiring TORC1 activity as a readout for a large number of individuals, it is necessary to have a high-throughput methodology that allows monitoring the activation of this pathway in numerous yeast strains. In this context, the present protocol was designed to assess phenotypical differences in TORC1 activation using a new reporter plasmid, the pTOMAN-G plasmid, specifically designed to monitor TORC1 activation. As a proof of concept, this methodology allowed phenotyping a large population of yeast strains derived from the 1002 Yeast Genomes Project, the most complete catalog of genetic variation in yeasts. This protocol proved to be an efficient alternative to assess TORC1 pathway activation compared to techniques based on immunoblot detection, which, although effective, are considerably more laborious. Briefly, the protocol involves the design and construction of the pTOMAN-G plasmid, which carries a construct containing the firefly luciferase gene (Luc) under the control of the TORC1-regulated RPL26A gene promoter (PRPL26A). The protocol then details the process for selecting subgroups of yeasts based on their ability to grow under nutrient-limited conditions, using proline as the sole nitrogen source. These yeasts are then transformed with the TOMAN-G plasmid, using two alternative transformation methods. Finally, those yeasts that emit luminescence are selected, whose phenotype for TORC1 activation is measured by a nitrogen-upshift experiment in microculture. This approach, using the pTOMAN-G plasmid, offers a rapid and consistent method for assessing TORC1 signaling pathway activation in a large number of yeast strains, highlighting its usefulness to study the activation of the TORC1 pathway and the domestication process associated with it. In the future, a redesign of the plasmid could extend its use as a reporter tool to monitor the activation of the TORC1 pathway, or other pathways, in other yeast species.

0 Q&A 577 Views Jun 20, 2025

Cancer-associated mesenchymal stem cells (Ca-MSCs), an integral part of the tumor microenvironment, play a major role in modulating tumor progression; they have been reported to progress as well as inhibit various cancers, including cervical cancer. To understand the exact role of Ca-MSCs in tumor modulation, it is necessary to have an optimized protocol for Ca-MSCs isolation. This work demonstrates the isolation and expansion of a primary culture of cervical cancer–associated MSCs (CCa-MSCs) from the biopsy sample of cervical cancer patients using the explant culture technique. The isolated cells were characterized according to International Society for Cellular Therapy (ISCT) guidelines. Morphological analysis revealed that cells were adherent to the plastic surface and possessed spindle-shaped morphology. Flow cytometry analysis of the cells showed high expression (~98%) for MSC-specific cell surface markers (CD90, CD73, and CD105), negative expression (<0.5%) for endothelial cell marker (CD34) and hematopoietic cell marker (CD45), and negligible expression for HLA-DR, as recommended by ISCT. Further, trilineage differentiation potential analysis of the cells showed their osteogenic and chondrogenic potential and adipogenic differentiation. This standardized protocol will assist in the cultivation of CCa-MSCs and the study of their interactions with tumor cells and other components of the tumor microenvironment. This protocol may be utilized in the establishment of Ca-MSCs from other types of cancers as well.