BioTechniques最新文献

Cell line stability of mammalian cell lines is an important concern for manufacturing of biopharmaceuticals. Characterization of the master cell bank (MCB) for monoclonal antibody A (Mab A) via multiplex fluorescence in situ hybridization (M-FISH) analysis identified two distinct karyotypic cell populations. As a result, a regulatory agency requested additional analysis to support a single clonal lineage of the MCB and demonstrate control of the two cell populations in subsequently derived cell banks. Next Generation Sequencing (NGS) revealed 11 transgene integration site sequences. A multiplex (measure two amplicons) Droplet Digital PCR (ddPCR) method was developed and characterized those 11 integration site copy numbers in the MCB. Analysis of 12 subclones from the MCB displayed that among 7 subclones there was loss of at least one of five specific integration sites. The characterization of these five unstable sites in the end production cells (EPCs) of four manufacturing batches are consistent with the MCB. The conservation of five other integration sites among all clones supported that these cells were derived from the same population and that loss of the unstable integration sites contributed to the two distinct karyotypic populations.

Processing methods that do not induce shape distortion are essential for the analysis of tissue morphology. Although the use of agarose-gelatin double embedding to reduce shape distortion has been suggested, the effect of matrix concentration has not been addressed. Therefore, combinations of agarose (1-3%) and gelatin (1-10%) were evaluated using multicellular spheroids without extracellular matrix as a model of mechanical fragility. In this study, a blend of 2% agarose and 5% gelatin effectively prevented distortion. This protocol can enhance the morphological analysis of delicate tissues.

Introduction: Quantitative assessment of lung tissue architecture is essential for evaluating disease progression in experimental models of acute lung injury (ALI). However, conventional methods for measuring alveolar septum thickness rely on manual procedures that are time-consuming, labor-intensive, and, observer-dependent, compromises comparability across studies and reproducibility in translational biomedical research and drug development.Method: We present a novel, fully automated approach based on two-dimensional spatial autocorrelation function (2D-ACF) analysis to quantify septum thickness from hematoxylin and eosin (H&E)-stained lung sections.

After validation with simulated data, the method was applied to a murine model of ALI induced by lipopolysaccharide (LPS) instillation under normal (ND) or high-fat diet (HFD) conditions.Results: The 2D-ACF provided an unbiased estimate of mean septum thickness across entire tissue images. LPS instillation increased thickness more than sixfold versus controls, with further enlargement in the HFD+Instilled group, capturing additive effects of metabolic stress across >400 images.

Manual measurements showed substantial inter-observer variability, especially in injured lungs, whereas the 2D-ACF was observer-independent and correlated strongly with traditional measurements in the instilled group (r = 0.89, p < 0.001).

Discussion: The 2D-ACF framework offers a reproducible, scalable, and unbiased tool for lung pathology studies and other tissues exhibiting spatial heterogeneity in histological architecture.

Method summary: Paraformaldehyde-fixed mouse liver samples are sectioned on a vibratome at 100 µm. The free-floating sections are permeabilized, followed by exposure to one of two heat-induced antigen retrieval methods selected based on the antigen of interest. Antigen retrieval is carried out in a mechanical stabilization apparatus developed here to prevent free-floating sections from deforming. The immunostaining is then carried out with noted modifications to previously established protocols.

Multidisciplinary abstract: Spatial organ biology widely relies on slicing tissues into one cell-thick sections. These studies have advanced our understanding of organ structure and function; yet, thin sections have been insufficient to investigate three-dimensional organization in complex organs. A more recent approach utilizes thicker sections, enabling visualization of volumetric tissue architecture. Currently, few protocols exist to strengthen immunofluorescence signal on thick tissue sections while maintaining tissue integrity, particularly for difficult antigens. Here, we developed a simple, affordable method that mechanically stabilizes thick tissue sections and preserves tissue integrity during heat-mediated antigen retrieval used to enhance antigen accessibility. This method widens the utility of thick tissue sections and facilitates holistic analysis of complex tissues, opening new possibilities for spatial organ research.

The Applied Biosystems Axiom Microbiome Array enables high-throughput detection of bacteria, archaea, viruses, protozoa, and fungi across multiple samples. However, its native software outputs are not compatible with common downstream analysis tools, requiring preprocessing. We identified a lack of open-source pipelines tailored to these outputs. To address this gap, we developed AxioParse, a Python-based pipeline built with the Dagster orchestration framework that automates data cleaning, taxonomic mapping, and formatting for downstream analysis. AxioParse reduces manual processing and generates datasets compatible with platforms such as QIIME2 and R, improving reproducibility and facilitating broader use of the Axiom Microbiome Array in microbiome research (https://github.com/Eghtesady-Lab-Bioinformatics/axioparse).

BackgroundLong single-stranded DNA (ssDNA; >200 nucleotides) is valuable for DNA nanotechnology, precision medicine, and as a CRISPR-Cas9 knock-in donor template, but existing preparation methods are laborious, low-yield, or difficult to scale. MethodsWe developed a workflow combining enzymatic digestion with high-temperature reversed-phase high-performance liquid chromatography (RP-HPLC) to purify kilobase-length ssDNA. The method was evaluated across analytical and semi-preparative formats. ResultsThe approach enables clean resolution of linear and circular ssDNA species ranging from 1.5 to 4.5 kb and is scalable across formats. A 1.5 kb ssDNA donor supported efficient CRISPR knock-in at the T-cell receptor alpha constant (TRAC) locus in primary human CD8⁺ T cells, without adversely affecting viability or expansion. ConclusionsThis RP-HPLC workflow provides a scalable and reproducible method for generating high-purity long ssDNA suitable for genome engineering applications.

Catalytic DNA molecules (DNAzymes) offer a programmable means to manipulate RNA without genomic modification. Although DNAzymes have been thoroughly characterized in culture and in various animals, their utility in the prevalent model system, Caenorhabditis elegans, has not been explored. Here, we examine the feasibility of catalytic DNA-mediated RNA targeting in C. elegans. Cholesterol-conjugated 8-17 DNAzymes were efficiently delivered to intestinal cells following ingestion, with no visibile toxic effects on growth or health, reducing targeted reporter fluorescence and endogenous gene function. The observed dependence on cholesterol tagging was consistent with a requirement for cellular uptake. Our findings provide a proof-of-principle demonstration of catalytic DNA activity in C. elegans, and highlight considerations for design and optimization. This exploratory study lays the foundation for expanding catalytic nucleic acid technologies in nematodes, supporting future development of programmable RNA tools in a powerful model system.

This study developed a detection method for antisense oligonucleotides (ASOs) by ligase detection reaction (LDR) using T4 RNA ligase 2 (T4Rnl2). The LDR was applied to detect a nusinersen-based ASO, an 18-mer 2'-O-methoxyethyl-modifiedoligonucleotide with phosphorothioate linkages. The method enabled detection of the ASO at concentrations of 0.08-10 nM in aqueous samples. In addition, when combined with mixed-mode solid phase extraction for ASO spiked samples, the method detected 0.8-100 nM in serum and 0.04-25 nmol/g tissue in spinal tissue. The method was further applied to determine ASO levels in plasma and spinal tissue following intrathecal injection in mice. These results demonstrated that LDR using T4Rnl2 was a simple and sensitive approach for detecting ASOs in biological samples.