STAR Protocols最新文献

Here, we present a protocol to study epidermal growth factor (EGF) receptor (EGFR) or transferrin trafficking in mammalian cells. We describe steps for using fluorescent ligands or antibodies, confocal imaging, and quantitative analysis to track their movement. We detail procedures for cell culture preparation, labeling membrane proteins, optimizing imaging, and isolating cell lysates for the biochemical analysis of EGFR degradation after EGF treatment. This protocol is adaptable to various cell types and for assessing genetic or pharmacological impacts on endosomal trafficking. For complete details on the use and execution of this protocol, please refer to Ye et al.,¹ Ye et al.,² and Wang et al.³.

Passive thermal management of an object can be achieved by manipulating its optical spectrum. Here, we present a protocol to create a dual-mode Janus fabric (DMJF) with unique switchable thermal management properties. We describe steps for fabricating radiative cooling (RC), ultraviolet (UV) reflective, and long-wave infrared (LWIR) shielding layers. We then detail steps for optical characterization and permeability and thermal measurements for the fabricated samples. This protocol could be applicable to various dynamic passive thermal management scenarios under diverse climatic conditions. For complete details on the use and execution of this protocol, please refer to Pian et al.¹.

TargetOrtho2 uses transcription factor binding site information to predict transcription factor targets in C. elegans, based on an in silico phylogenetic footprinting approach. Here, we present a protocol to identify transcription factor target genes using a new version of TargetOrtho2. We provide instructions for installing TargetOrtho2 and its required suite of programs, for predicting transcription factor target genes, and for updating and adding new genomes to TargetOrtho2.

Preprocessing is a critical yet challenging step in electroencephalography (EEG) research due to its significant potential impact on results. We present a protocol for semi-automatic EEG preprocessing incorporating independent component analysis (ICA) and principal component analysis (PCA) with step-by-step quality checking to ensure removal of large-amplitude artifacts. We describe steps for interpolating bad channels, removal of major artifacts by ICA and PCA correction, and exporting processed data. This protocol produced consistent results from users with a broad range of experience.

Trichoderma koningiopsis is a filamentous fungus that produces numerous bioactive molecules and has the potential to be used as a biological control and plant growth-promoting agent. Here, we present a protocol for generating protoplasts of T. koningiopsis. We then describe four techniques for the stable transformation of plasmid DNA: polyethylene glycol (PEG)-mediated transformation of protoplasts, Agrobacterium-mediated transformation, electroporation of spores, and lipofection of mycelia. We then detail procedures for confirming the expression of a spCas9 gene using fluorescence microscopy.

Lung epithelial cilia play a crucial role in respiratory health by driving mucociliary transport (MCT) and clearing inhaled pathogens and materials. Here, we present a protocol for measuring both ciliary beat frequency (CBF) and MCT in mucociliary human small airway epithelial cells. We describe steps for configuring experimental setups, collecting CBF and MCT data, analyzing datasets, and performing post-acquisition analysis. This protocol is adaptable to mucociliary cells from other lung regions and to mouse tracheal epithelial cells. For complete details on the use and execution of this protocol, please refer to Thomas et al.¹.

The lack of standardized techniques for processing complex health data from COVID-19 patients hinders the development of accurate predictive models in healthcare. To address this, we present a protocol for utilizing real-world multivariate time-series electronic health records of COVID-19 patients. We describe steps for covering the necessary setup, data standardization, and formatting. We then provide detailed instructions for creating datasets and for training and evaluating AI models designed to predict two key outcomes: in-hospital mortality and length of stay. For complete details on the use and execution of this protocol, please refer to Gao et al.¹.

To unravel the complexity of biological processes, it is necessary to resolve the underlying protein organization down to single proteins. Here, we present a protocol for secondary label-based unlimited multiplexed DNA-PAINT (SUM-PAINT), a DNA-PAINT-based super-resolution microscopy technique that is capable of resolving virtually unlimited protein species with single-protein resolution. We describe the steps to prepare neuronal cultures, troubleshoot and conduct SUM-PAINT experiments, and analyze the resulting feature-rich neuronal cell atlases using unsupervised machine learning approaches. For complete details on the use and execution of this protocol, please refer to Unterauer et al.¹.

Pulmonary alveolar epithelial type I (AT1) cells have a flattened morphology to permit the diffusion of oxygen into the capillaries and historically have been difficult to isolate or maintain in culture. Here, we present a protocol for generating human alveolar type I-like cells (induced pluripotent stem cell-derived AT1s [iAT1s]) from induced pluripotent stem cell-derived alveolar epithelial type II cells (iAT2s) in vitro. We describe steps to plate iAT1s in either 3D or air-liquid interface cultures and to analyze or isolate iAT1s via flow cytometry. For complete details on the use and execution of this protocol, please refer to Burgess et al.¹.

Mutations at RNA splicing sites or regulatory elements can alter splicing efficiency or patterns, affecting RNA functionality and tissue-specific expression. Here, we present a protocol to study the impact of mutations near splicing sites on precursor mRNA (pre-mRNA) splicing. We describe steps for constructing plasmids by cloning the target gene into the pEGFP-N1 vector, performing site-directed mutagenesis, and transiently transfecting HEK293 cells. We then detail procedures for conducting mini-gene splicing assays to analyze splicing patterns influenced by mutations. For complete details on the use and execution of this protocol, please refer to Peng et al.¹.