Efficient purification of extracellular vesicles via circular multicavity electrophoresis coupled with ultrafiltration.

Rapid discovery of cell-surface glycosylation regulators using a lectin-based magnetic CRISPR screen.

Astrocytic activation is critically involved in the development and maintenance of bone cancer pain (BCP). Recent studies have shown that astrocytes participate in synaptic remodeling through synaptic phagocytosis, whereas neuronal cell adhesion molecule (NRCAM) restricts the extension of perisynaptic astrocytic processes and exerts an anti-phagocytic effect. However, whether astrocyte-mediated synaptic phagocytosis contributes to BCP remains to be explored. Hence, this study investigated the role of astrocytic NRCAM deficiency in synaptic remodeling and central sensitization in BCP. A mouse model of BCP was established by intrafemoral inoculation of fibrosarcoma cells. Pain-related behaviors were evaluated by spontaneous pain assessment and mechanical withdrawal threshold testing. Bone destruction and tumor infiltration were examined by hematoxylin and eosin staining. Synaptic alterations and astrocytic activation were assessed by western blotting and immunofluorescence. Astrocyte-mediated synaptic engulfment was analyzed using immunofluorescence, three-dimensional reconstruction, magnetic-activated cell sorting, and Golgi-Cox staining. Moreover, fluorocitrate was used to inhibit the astrocytic activation. Astrocytic NRCAM was overexpressed via adeno-associated virus (AAV)-mediated gene delivery. Fibrosarcoma cell inoculation induced persistent spontaneous pain and mechanical hyperalgesia in C3H/HeN mice, accompanied by trabecular bone destruction and tumor infiltration. In the spinal cord, expression of the excitatory synaptic markers VGLUT1 and PSD95 was increased, whereas expression of the inhibitory synaptic markers VGAT and Gephyrin was decreased, together with marked astrocytic activation. Multiple complementary assays consistently showed that astrocytes engulfed both excitatory and inhibitory synapses, with a clear preference for GABAergic synapses. Pharmacological inhibition of astrocytic activation with fluorocitrate alleviated pain behaviors and improved synaptic remodeling. NRCAM expression was significantly downregulated in tumor-bearing mice. Restoration of astrocytic NRCAM expression by AAV markedly reduced astrocyte-mediated engulfment towards GABAergic synapses, had minimal effect on excitatory synapses, and significantly attenuated BCP. Reactive astrocytes preferentially phagocytose GABAergic synapses in BCP, thereby contributing to synaptic imbalance and central sensitization. This process is associated with downregulation of astrocytic NRCAM. Restoring astrocytic NRCAM alleviates BCP by suppressing excessive astrocyte-mediated phagocytosis of GABAergic synapses. These findings identify astrocytic NRCAM-dependent synaptic phagocytosis as an unrecognized mechanism underlying BCP and as a potential therapeutic target.

Protocol for isolating and culturing microglia from the adult mouse brain using a magnetic-activated cell sorting system.

Protocol for isolating and culturing human monocytes from peripheral blood and measuring intracellular calcium dynamics using Fura-2.

We describe a method for purifying murine cortex mitochondria, based on a modification of available technology. The mitochondria isolated and purified using this modified method are viable, without plasma membrane contamination, and suitable for downstream analyses. The method is based on magnetic-activated cell sorting (MACS) technology: extracting mitochondria using a commercially available kit, followed by isolation of mitochondria using antibody-labeled magnetic beads and additional washing steps. We identify the steps crucial for purification from plasma membrane contamination, including optimal starting tissue weight, essential reagents and their preparation, as well as other experimental conditions. The viability of purified mitochondria is assessed using fluorescent imaging. Furthermore, we compare the purity using two additional suboptimal methods in regards to plasma membrane contamination: one that employs an additional commercial kit and protocol, and another that is based on differential centrifugation. The method of choice results in a lower mitochondrial yield, but maximal purity in respect to copurification with other cellular membranes, which is especially important for downstream analyses of high sensitivity. • Successfully overcoming the notoriously challenging step of purification mitochondria free from plasma membrane contamination. • Purity is also achieved in respect to other cellular organelle contamination. • Viable mitochondria is obtained, suitable for downstream analyses.

Regenerative potential of muse cells in ROS-mediated cardiac injury: An in vitro oxygen-glucose deprivation/reoxygenation model.

Exploring fetal cells in maternal blood in relation to rupture of the fetal membranes.

Advances in rodent oligodendrocyte precursor cells isolation and culture: From traditional methods to modern approaches.

Glioblastoma (GBM) is the most aggressive primary malignant brain tumor and is associated with limited treatment options and poor prognosis. Conventional GBM cell lines undergo genetic drift and progressive divergence from patient-relevant molecular features over long-term culture, limiting their translational relevance. This study aimed to establish and characterize a novel isocitrate dehydrogenase 1 (IDH1)-wildtype GBM cell line, SHG142, along with its associated glioma stem-like cells (GSCs) and organoid models, to advance GBM biology and therapeutic research. Primary tumor tissue from a 70-year-old female patient was cultured under standard conditions to generate the SHG142 cell line, which was authenticated by short tandem repeat (STR) profiling. Phenotypic and genetic features were evaluated using immunofluorescence (IF), karyotyping, and whole-exome sequencing (WES) including a concordance analysis between an early bulk primary culture (P2) and late-passage SHG142 (P50). GSCs were isolated via serum-free culture and magnetic-activated cell sorting (MACS). Tumorigenicity and invasiveness were assessed through intracranial xenografts in nude mice (n = 6/group) and coculture with brain organoids derived from human embryonic stem cells (n = 4 independent organoids). SHG142 cells exhibited stable morphology and proliferation over more than 50 passages, retained key GBM markers (e.g., Nestin, Glial Fibrillary Acidic Protein (GFAP), O6-methylguanine-DNA methyltransferase (MGMT), Ki-67, P53, S-100, CD34), and exhibited chromosomal instability (+7, -10, and t(9;13)). WES revealed mutations in the TERT promoter, TP53 and NF1 and supported lineage continuity from P2 to P50 through shared SNVs and conserved driver events. CD133+ SHG142 GSCs demonstrated stem-like properties and invasive behavior in vivo and in organoid coculture. Compared with xenografts derived from SHG142 cells, GSC-derived xenografts were associated with increased malignancy and shorter survival (median survival 25 vs. 31.5 days, p = 0.025). The organoid coculture model confirmed deep infiltration by proliferative, stem-like tumor cells. SHG142 and its GSCs represent a phenotypically stable, genetically defined GBM model with evidence of lineage continuity and retention of key driver events during in vitro propagation. Their tumorigenic and invasive properties make them valuable tools for mechanistic studies and preclinical therapeutic screening, supported by initial two-dimensional and three-dimensional drug response readouts that enable first-pass evaluation of therapeutic response.

The Escherichia coli hitchhiker co-translocation mechanism has been adapted as a screening platform known as Functional Ligand-binding Identification by Tat-based Recognition of Associating Proteins (FLI-TRAP) for identifying and isolating interacting proteins. Traditional FLI-TRAP systems utilize either monocistronic or bicistronic expression constructs. However, in the context of selecting single-domain antibody fragments, or ‘nanobodies’ (Nbs), specific to bovine interferon-gamma (bIFN-γ), the monocistronic system produces insufficient expression levels while the original bicistronic system is prone to false positives due to potential structural rearrangements that allow antibiotic resistance independent of Nb–antigen binding. To overcome these limitations, we have modified an improved bicistronic FLI-TRAP system that reduces the rate of false positives and enhances the fidelity of the selection process. This optimized system was validated through isolating anti-bIFN-γ Nbs from a fully synthetic yeast display library. Candidate Nbs were first enriched using magnetic-activated cell sorting (MACS), followed by screening via the improved FLI-TRAP assay. The two best clones, B7 and N5, displayed specific binding to bIFN-γ, with dissociation constants (KD) of 81.6 nM and 575 nM, respectively. Further characterization revealed that B7 exhibited high specificity, low polyreactivity, and effective detection of bIFN-γ in bovine plasma samples, compared with the BOVIGAM kit, a commercially approved and widely used diagnostic tool for bovine tuberculosis. This study demonstrates the utility of the improved bicistronic FLI-TRAP system as a reliable, scalable, and cost-effective platform for selecting high-affinity Nbs. The system offers a promising alternative for developing Nb-based diagnostic kits, which are particularly suited for use in resource-limited settings.

An effective method for recovering rat spermatogonial stem cells from frozen rat testis cells.

Umbilical cord blood (CB)-derived natural killer (NK(CB)) cells are a promising immunotherapeutic modality due to their cytotoxicity, allogeneic compatibility, and reduced immunogenicity relative to peripheral blood NK cells (NK(PB)). Although several commercial culture media have been optimized for NK(PB) expansion, their suitability for NK(CB) cells remains insufficiently characterized. This study compares the expansion efficiency, phenotypic shifts, functional activation, and cost-effectiveness of NK(CB) cells expanded ex vivo under feeder-free static culture conditions with IL-2-supplemented culture media. CB-derived CD56⁺ cells isolated by magnetic-activated cell sorting were cultured in six candidate culture media, after which CTS™ NK-Xpander™ (NKX) and NK MACS® (NKM) with 1% (NKM1) or 2% (NKM2) supplement were selected for further evaluation. NKX achieved the highest average fold expansion (18.8 ± 7.0), followed by NKM2 (16.8 ± 7.3) and NKM1 (14.3 ± 3.5). All conditions increased cytotoxicity and CD107a degranulation, with NKX consistently yielding stronger functional responses. Phenotypic analysis revealed a shift toward a CD56brightCD16⁻ profile, particularly in NKM cultures, accompanied by cytokine-associated CD16 downregulation. Cost analysis identified NKX and NKM1 as the most cost-efficient conditions, whereas NKM2 doubled manufacturing costs due to higher supplement requirements. In conclusion, NKX and NKM media effectively support feeder-free ex vivo expansion of NK(CB) cells with high viability and robust functional activation. Phenotypic changes, donor-dependent variability, and CD16 downregulation should be considered when designing clinically translatable manufacturing strategies. These findings are essential to enhance scalability, reduce manufacturing costs, and advance NK(CB) cells as an accessible and effective immunotherapy, facilitating their integration into clinical-grade manufacturing and scalable bioprocessing pipelines.

Dysregulated mitochondrial DNA (mtDNA) promotes inflammatory response and disease progression. However, the mechanism and role of mtDNA-mediated inflammatory activation in the pathogenesis of Parkinson's disease (PD) are not yet clear. This study demonstrates that the injection of mtDNA into the substantia nigra pars compacta induces PD pathology in mice, characterized by the loss of dopaminergic (DA) neurons and the activation of microglia. Transcriptomic profiling of magnetic-activated cell sorting (MACS)-sorted cells reveals a pronounced upregulation of genes associated with the NLRP3 inflammasome pathway in microglia following the mtDNA administration. Critically, lipopolysaccharide (LPS) and rotenone induced in vivo and in vitro PD models show oxidized mtDNA (ox-mtDNA) release and microglial NLRP3-IL-1β axis activation as evidenced by upregulation of NLRP3 and IL-1β, caspase-1 cleavage, and IL-1β release. The role of mtDNA in activating the NLRP3-IL-1β axis is further validated in BV2 cells through exogeneous mtDNA transfection, while the NLRP3-IL-1β activation is negated in the LPS and rotenone induced model when mtDNA release is inhibited. Especially, oxidized mtDNA is superior to nonoxidized mtDNA in activating the NLRP3-IL-1β axis. NLRP3 knockdown in BV2 cells abolishes the activation of NLRP3-IL-1β axis induced by mtDNA or exposure of LPS and rotenone and mitigates the damage to SH-SY5Y cells in co-culture systems. Ox-mtDNA-mediated neuronal cell damage is initiated through binding to NLRP3, as demonstrated by co-immunoprecipitation and co-localization in BV2 cells. Molecular docking prediction and analysis of intrinsically disordered region (IDR) of NLRP3 indicate that ox-mtDNA interacts with the positively charged IDR of NLRP3. This interaction is validated by electrophoretic mobility shift and in vitro PYD-caspase-1 cleavage assays, demonstrating the formation of the ox-mtDNA-NLRP3 complex and subsequent activation of NLRP3. This study describes a critical role of mtDNA in activating microglial NLRP3-IL-1β axis, leading to neurodegeneration in PD pathology, which provides clear clues for developing anti-PD drugs targeting NLRP3.

Immune cell separation for cell therapy: A comprehensive review of techniques, and challenges.

Efficiency of magnetic-activated cell sorting using Y-specific monoclonal antibodies on the quality of porcine sexed semen.

Sperm DNA fragmentation is a recognized factor in male infertility with direct implications for embryo development, implantation and pregnancy outcomes. Historically, standard semen analysis has not included assessments of DNA integrity, creating a clear need for advanced diagnostic tools. DNA damage can arise through pathways such as apoptosis, oxidative stress and exposure to environmental toxins, all of which compromise reproductive potential. Several methodologies exist for measuring spermDNA fragmentation, including the sperm chromatin structure assay, terminal deoxynucleotidyl transferase dUTP nick end labelling(TUNEL), the Comet assay and sperm chromatin dispersion, each of which has unique advantages and limitations. Novel automated imaging platforms incorporating machine learning algorithms have emerged, enabling high-throughput, single-sperm assessment and reducing subjectivity associated with manual scoring. Species differences further complicate the understanding of DNA stability and sperm quality, especially in livestock and models of artificial insemination; nonetheless, physiological similarities between humans and species close to humans provide useful translational insights. Emerging sperm selection technologies, including microfluidics, hyaluronic acid affinity systems and magnetic-activated cell sorting, show promise in reducing DNA fragmentation, improving reproductive outcomes and decreasing pregnancy loss. As the field progresses toward increasingly personalized fertility treatments, measures of DNA integrity will remain central to optimizing assisted reproduction success rates across species.

Although protein–protein interactions (PPIs) are central to nearly all biological processes, identifying and engineering high-affinity intracellular binders remains a significant challenge due to the complexity of the cellular environment and the folding constraints of proteins. Here, we present a two-stage complementary platform that combines magnetic-activated cell sorting (MACS)-based yeast surface display with functional ligand-binding identification by twin-arginine translocation (Tat)-based recognition of associating proteins (FLI-TRAP), a bacterial genetic selection system for efficient screening, validation, and optimization of PPIs. In the first stage, MACS-based yeast display enables the rapid high-throughput identification of candidate binders for a target antigen from a large synthetic-yeast display library through extracellular interaction screening. In the second stage, an antigen-focused library is subcloned into the FLI-TRAP system, which exploits the hitchhiker export process of the Escherichia coli Tat pathway to evaluate binder–antigen binding in the cytoplasm. This stage is achieved by co-expressing a Tat signal peptide–tagged protein of interest with a β-lactamase-tagged antigen target, such that only binder–antigen pairs with sufficient affinity are co-translocated into the periplasm, thus rendering the bacterium β-lactam antibiotic resistant. Because Tat-dependent export requires fully folded and soluble proteins, FLI-TRAP further serves as a stringent in vivo filter for intracellular compatibility, folding, and stability. Therefore, this approach provides a powerful and cost-effective pipeline for discovering and engineering intracellular protein binders with high affinity, specificity, and functional expression in bacterial systems. This workflow holds promise for several applications, including synthetic biology and screening of theragnostic proteins and PPI inhibitors.Key features• Combines a single round of MACS enrichment with FLI-TRAP for high-throughput Nb discovery.• Reduces time and resource demands compared to traditional workflows involving multiple rounds of MACS/FACS.• Enables in vivo selection of high-affinity, functional binders via Tat-dependent export linked to β-lactam resistance, correlating binding affinity and solubility with antibiotic resistance.

BackgroundWhile immunoblobulin A(IgA) dominates gut mucosal immunity, the roles of immunoglobulin M (IgM) and immunoglobulin G (IgG) in host-microbiota interactions remain poorly characterized, particularly in schizophrenia (SCZ). Although gut dysbiosis and immune activation have been implicated in SCZ,the contribution of IgG/IgM-coated gut microbiota to disease associated inflammation and behavioral alterations remains unknown.MethodsWe recruited six patients with SCZ, six with other psychiatric disorders (OPD) and six age- and sex- matched healthy controls. IgG/IgM-coated gut microbiota were isolated from 100 mg fecal samples via magnetic-activated cell sorting (MACS) and profiled by 16S rRNA sequencing. A pilot an IgG/IgM-coated fecal microbiota transplantation (FMT) using anaerobically cultured human intestinal microbiota was conducted in mice to assess the effects on gut pathology, peripheral immunity, and behavior. The percentage of neutrophil granulocyte in peripheral blood was quantified microscopically, and statistical analyses were performed using one-way ANOVA in GraphPad Prism 8, with (p < 0.05.ResultsThe proportions of IgM-coated bacteria was significantly higher in patients with SCZ than in healthy controls (p<0.05), with enrichment of Rhodococcuss, Shigella, Clostridium and Streptococcus. Mice receiving a mixture of high-IgM-coated intestinal bacteria mixture showed reduced depletion of peripheral neutrophils, mild colon shortening, and mucosal inflammation compared with those receiving low IgM-coated or uncoated bacteria. In contrast, high IgG-coated bacteria, enriched in Rhodococcuss, Acinetobater and Pseudomonas, decreased in SCZ, but induced similar inflammatory gut changes. No IgG- nor IgM- induced anxiety-like behavior were detect in the mice.ConclusionsOur findings reveal that IgG/IgM-coated intestinal microbiota display distinct immunoreactive microbiota signatures associated with SCZ. These coated communities promote gut inflammation without inducing anxiety-like behavior, highlighting their potential as novel biomarkers of SCZ-associated immune dysregulation and as targets for personalized therapeutic strategies.

ABSTRACTStem cell-derived β-cells (SCβ-cell) are a renewable and scalable alternative to cadaveric islets as a cell-replacement therapy for type 1 diabetes (T1D). However, heterogeneity within SCβ-cell cultures remains problematic for graft safety and function. Magnetic selection of SCβ-cells expressing a unique cell-surface marker may help deplete undesirable cell types and facilitate functional maturation. Here, we explored the transmembrane glycoprotein CD19 as a potential cell-surface marker for the enrichment of insulin-expressing SCβ-cells. Using CRISPR/Cas9 technology, we created a knock-in add-on of CD19-mScarlet downstream of insulin (INS) coding sequence exon 2 in human embryonic stem cells (hESCs). We developed and optimized a magnetic-activated cell sorting protocol for CD19-mScarlet-expressing cells, forming enriched SCβ-cell clusters with improved glucose-stimulated C-peptide secretion. This strategy holds promise to facilitate large-scale production of functional SCβ-cells for disease modeling and cell-replacement therapy.