STAR Protocols最新文献

Bacteriocytes are specialized insect cells adapted to harbor symbionts. However, their low number in individual whiteflies makes obtaining enough for transcriptome sequencing challenging. Here, we present a protocol for the isolation of whitefly bacteriocytes. We describe steps for preparing the single bacteriocyte sample, cDNA amplification library construction, sequencing, and data analysis. Our protocol offers convenience and efficiency for conducting single-cell sequencing for bacteriocytes and has the potential to be applied to other insect cells. For complete details on the use and execution of this protocol, please refer to Li et al.¹.

Here, we present a protocol for cell-type-specific single-cell labeling and manipulation in Drosophila using a sparse driver system. We describe steps for generating constructs and fly lines, titrating heat-shocked durations for precise temporal control and desired sparsity, and co-expressing multiple transgenes for experiments. We demonstrate that this generalizable toolkit enables tunable sparsity, multi-color staining, single-cell trans-synaptic tracing, single-cell manipulation, and cell-autonomous gene function analysis. For complete details on the use and execution of this protocol, please refer to Xu et al.¹.

When investigating cellular responses to viral infections, there is a need to quantitatively distinguish between dying cells, infected cells, and cells that are both infected and dying. Here, we present a protocol to simultaneously detect porcine reproductive and respiratory syndrome virus (PRRSV) infection and cell death using a flow cytometry double-staining approach. We describe steps for cell seeding, infection, and collection; staining dead cells; sample fixation; and cell permeabilization. We then detail procedures for immunostaining and flow cytometry analysis.

Human cerebral organoids (hCOs) provide an excellent model for the study of human brain development and disease. Here, we present a protocol to obtain hCOs directly from two-dimensional (2D) pluripotent stem cell (PSC) cultures, avoiding cell dissociation and posterior embryoid body (EB) aggregation. We describe steps for subjecting 2D cultures to a neural fate and subsequently developing hCOs. We then detail the evaluation of different cellular types. For complete details on the use and execution of this protocol, please refer to González-Sastre et al.¹.

Colostrum and milk from dairy sources consist of whey, casein, and fat, which have notable pharmacological properties due to their proteins and peptides. Here, we present a protocol for isolating, simulating in vitro gastrointestinal digestion, and fractionating colostrum and milk hydrolysates from any dairy source. We also describe steps for nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nano-LC-ESI-MS/MS) identification of proteins and peptides and in silico system biology-based profiling of the proteins and peptides present in the hydrolysates.

T cell-based therapies hold immense promise, but their production remains time-consuming and technically complex. Here, we present a protocol that streamlines the activation and lentiviral transduction of primary human T cells with artificial receptors. We describe steps for T cell isolation, lentivirus production, and the simultaneous activation/transduction of T cells. By eliminating magnetic T cell activation beads, sequential steps, and lengthy spinoculation, this protocol significantly enhances efficiency, scalability, and accessibility for research and therapeutic applications.

Mechanical testing of battery components is essential for understanding their mechanical behavior and developing accurate numerical models. Here, we present a protocol for characterizing the mechanical properties of lithium-ion batteries. We describe steps for performing tests on compression, tension, and indentation to evaluate the individual components. Furthermore, we detail procedures for compression testing on stacked structures to assess the mechanical properties of the battery jelly roll, providing key insights into its performance under real-world conditions. For complete details on the use and execution of this protocol, please refer to Wang et al.¹.

Single-cell RNA sequencing provides a deep understanding of gene expression patterns at the single-cell level, enabling researchers to study cellular heterogeneity and differentiation. Spatial transcriptomics, meanwhile, delivers spatial visualization and quantitative analysis of tissue sections. Here, we introduce a protocol to isolate single cells and obtain high-quality spatial transcriptomics data from bladder Ewing sarcoma tissue. We outline steps for sample collection, single-cell RNA sequencing, and spatial transcriptomics sequencing. For complete details on the use and execution of this protocol, please refer to Mao et al.¹.

Cytometry by time of flight (CyTOF) is a flow cytometry-based technique using metal-tagged antibodies, allowing immunophenotyping. Here, we present a protocol for phenotyping mouse myeloid and lymphoid cells isolated from lung, spleen, and intraperitoneal lavages in healthy or pathologic conditions. We describe steps for antibody labeling and titration, tissue dissociations, and staining. We then detail procedures to compare staining obtained from fresh or cryopreserved tissues. For complete details on the use and execution of this protocol, please refer to Morin et al.¹.

Here, we present a protocol to perform a time-kill assay (TKA) to quantify bacterial burden at multiple time points using colony-forming units and most probable number readouts simultaneously. We describe steps for preparing inoculum, experimental conditions, and sampling bacterial counts. We then detail procedures for quantification and analysis. TKAs provide longitudinal data reflecting the dynamics of the antibiotic effect over time against a planktonic culture and quantify the concentration-effect relationship. For complete details on the use and execution of this protocol, please refer to Van Wijk et al.¹.