Methods and Protocols

Telomere length is a crucial marker of cellular aging and genomic stability, with significant implications for age-related diseases and cancers. This study introduces an improved quantitative PCR (qPCR) method for measuring absolute telomere length, addressing the need for accurate and high-throughput assessment in both clinical and research settings. Novel primers were designed for the single-copy gene interferon beta (IFNB1) to serve as an internal control, alongside a series of single-stranded oligonucleotide standards to establish a calibration curve. This approach allows for precise quantification of telomere length in kilobases per single copy gene copy number per chromosome. We validated this method using DNA samples from peripheral blood and buccal swabs from 17 healthy human volunteers, as well as umbilical cord blood from 9 healthy newborn babies, demonstrating its high linearity and reproducibility. Our findings indicate that this improved qPCR technique provides a rapid, cost-effective, and accurate means of measuring absolute telomere length, thereby facilitating large-scale studies and enhancing clinical diagnostics related to telomere biology.

Three-dimensional (3D) culture systems that recapitulate the tumor microenvironment are essential for studying cancer cell behavior, drug response, and cell–matrix interactions. Here, we present a detailed protocol for generating 3D spheroid cultures from murine breast cancer cells using methacrylated gelatin (GelMA)-based bioink and a CELLINK BioX bioprinter. This method enables precise deposition of spheroid-laden GelMA droplets into low-attachment plates, facilitating high-throughput and reproducible 3D culture formation. The protocol includes steps for spheroid formation, GelMA preparation, bioprinting, and post-printing analysis using a customized CellProfiler pipeline. The analysis pipeline takes advantage of the functionality of CellProfiler and ImageJ software (version 2.14.0) packages to create a versatile and accessible analysis tool. This approach provides a robust and adaptable platform for in vitro cancer research, including studies of metastasis, drug resistance, cancer cell lipid metabolism, and TME-associated hypoxia.

Rosin, a renewable natural resin derived from pine trees, is a promising biomass material for sustainable product development, though its distinct intrinsic odor limits broader use. This study implemented a comprehensive analytical strategy to mitigate the odor by incorporating essential oils (EOs)—eucalyptus (EUC) and peppermint (MINT)—and to conduct a multi-analytical characterization of the modified rosin jewelry. By integrating complementary analytical techniques, including LC-Q/TOF-MS for non-volatile components and GC-Q/TOF-MS for volatile organic compounds (VOCs), we achieved a systematic chemical profiling of the materials. The core composition of rosin, dominated by abietic acid (>48%), remained stable across all samples. The incorporation of EOs significantly altered the VOC profiles: The total VOC signal (summed peak area) in MINT-modified rosin was 2.57-fold that of the EUC-modified sample, with monoterpenoids comprising 87.62% of its VOC signature. Eucalyptol and limonene were tentatively identified as the major components in the EUC sample, whereas menthone, menthol, and limonene predominated in the MINT sample. Multivariate statistical analysis highlighted that variations in specific VOCs—particularly menthone, menthol, eucalyptol, and allo-ocimene—were closely associated with differences in the scent profiles of each modification. This work illustrates how a multi-technique analytical strategy can both guide and assess the functional modification of sustainable biomass materials. The findings offer a practical approach to improving rosin’s functional properties while providing a methodological framework for the integrated characterization of complex biomaterials, supporting the development of eco-friendly products aligned with green chemistry and sustainable design principles.