STAR Protocols最新文献

Ex vivo electroretinography (ERG) provides insight into the health and functionality of retinal cells, the integrity of phototransduction, and the visual cycle and allows for the direct application of pharmaceuticals to the retinal tissues. Here, we present a protocol for performing ex vivo ERG on adult zebrafish. We describe steps for zebrafish tissue dissection, mounting tissues, and assembling and connecting cassettes. We then detail procedures for running the Diagnosys software and processing and analyzing the resulting data.

Here, we present a protocol for guiding tissue preparation and flow cytometric analysis in subcutaneous murine tumor models and secondary lymphoid organs. We describe steps for dissociating tumors, spleens, and lymph nodes to obtain single-cell suspensions. We then detail procedures for immune cell staining and analysis and gating strategies including the use of fluorescence-minus-one controls (FMOs). This approach provides valuable insights into the impact of cancer therapies on the tumor and systemic immune response. For complete details on the use and execution of this protocol, please refer to Ingelshed et al.¹.

Single-cell RNA sequencing (scRNA-seq) enables detailed characterization of cell states but often lacks insights into tissue clonal structures. Here, we present a protocol to probe cell states and clonal information simultaneously by enriching mitochondrial DNA (mtDNA) variants from 3'-barcoded full-length cDNA. We describe steps for input library preparation, mtDNA enrichment, PCR product cleanup, and paired-end sequencing. We then detail computational steps for running maegatk, variant calling, and data integration to illuminate cell states and clonal dynamics in primary human tissues. For complete details on the use and execution of this protocol, please refer to Miller et al.¹.

Binding and neutralizing antibodies are critical indicators of protection against viral pathogens and are essential for assessing the immunogenicity and efficacy of a vaccine. Here, we present a protocol comprising two assays for measuring the spike-specific binding and neutralizing antibodies in mouse plasma following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination. We describe steps for determining binding antibody titers using enzyme-linked immunosorbent assay (ELISA) and assessing neutralizing antibody titers through a pseudovirus neutralization assay. For complete details on the use and execution of this protocol, please refer to Jingyou Yu et al.¹.

White adipose tissue (WAT) beiging holds significant therapeutic potential for combating obesity. Here, we present a protocol for inducing beige WAT in mice using both cold exposure and CL316,243 treatment. We describe steps for intraperitoneal injection, and subcutaneous WAT (sWAT) isolation, dissection, and fixation. We then detail procedures for histology, whole-mount immunofluorescence (IF) staining, and extracting RNA and protein. This protocol can be used for subsequent analysis to explore the mechanisms governing beige WAT induction in experimental settings, particularly the evaluation of angiogenesis. For complete details on the use and execution of this protocol, please refer to Wang et al.¹.

Extracellular vesicles (EVs) play a key role in cancer development and cellular homeostasis by transferring the biological cargo to recipient cells. Here, we describe steps for screening EV secretion-related genes by combining a microRNA (miRNA) library and ExoScreen, a highly sensitive EV detection technique. We also detail procedures for screening the direct target genes regulated by miRNAs. This protocol provides a useful tool for understanding complex intracellular communications involved in EV secretion. For complete details on the use and execution of this protocol, please refer to Yamamoto et al.¹.

Bone marrow stromal cells (BMSCs) serve as a valuable reservoir of multipotent stem cells important in the regulation of bone homeostasis and energy metabolism. Here, we present a protocol for isolating human BMSCs (hBMSCs) and characterizing their cellular metabolism related to hBMSC functional properties. We describe steps for bioenergetics, cell senescence, and production of reactive oxygen species (ROS), together with description of the data analysis. These assays provide information on hBMSC metabolic status valuable to regenerative medicine and therapeutic applications. For complete details on the use and execution of this protocol, please refer to Tencerova et al.¹.

The silk glands (SGs) of silkworms specifically synthesize silk proteins, thus strongly influencing the yield and quality of silk. Here, we present a protocol for isolating SG nuclei from silkworms and obtaining high-quality tissue slices for spatial transcriptomics. We describe steps for rearing, dissecting, and nucleus isolation. We then detail procedures for embedding, frozen section, and RNA capturing and sequencing. This protocol enables the exploration of the spatial distribution of SG cells at single-cell resolution. For complete details on the use and execution of this protocol, please refer to Ma et al.¹.

Liver sinusoidal endothelial cells (LSECs) line the liver sinusoids and play a crucial role in liver function. Isolating LSECs is beneficial for their functional evaluation in vitro. Here, we provide a protocol for obtaining purified LSECs from mice via gradient centrifugation and magnetic cell sorting (MACS), yielding cells suitable for culture and downstream analyses. We describe steps for culturing the purified LSECs and demonstrate their evaluation by flow cytometry. For complete details on the use and execution of this protocol, please refer to Papaioannou et al.¹.

Studies of human induced pluripotent stem cell (iPSC)-derived neurons promise important insights into neurodegenerative diseases. Here, we present a protocol for live imaging of axonal transport in glutamatergic iPSC-derived neurons (iNeurons). We describe steps for the differentiation of iPSCs into iNeurons via PiggyBac-mediated neurogenin 2 (NGN2) delivery, iNeuron culture and transfection, and the acquisition and analysis of time-lapse images. Our protocol is optimized for the widely available catalog of KOLF2.1J iPSCs with mutations relevant to neurodegenerative diseases but is also applicable to other iPSC lines. For complete details on the use and execution of this protocol, please refer to Dou et al.^1,2.