STAR Protocols最新文献

Chimeric antigen receptor (CAR) T cell therapy often causes serious toxicities, such as cytokine release syndrome (CRS), mainly due to interleukin-6 (IL-6) secreted by monocyte lineage cells. Here, we describe a protocol to generate anti-CD19 CAR T cells and quantify human monocyte-derived IL-6 cocultured with CAR T cells and target tumor cells in vitro. We further describe a protocol to generate a humanized mouse model and evaluate CAR T cell-associated plasma IL-6 levels in vivo. For complete details on the use and execution of this protocol, please refer to Yoshikawa et al.¹.

Here, we present a protocol for using d-rG4-seq, a technique for mapping RNA G-quadruplex (rG4) for chromatin-bound RNA. We describe steps for identifying in vivo rG4 structures based on differential sensitivity of rG4 to dimethyl sulfate (DMS) modification, folding in the presence of monovalent cations, K+ versus Li+, and reverse transcriptase (RT) readthrough when folded. We then detail procedures for isolating RNA from the chromatin-bound fractions to enrich for epigenetic regulators and comparing in vitro versus in vivo profiles. For complete details on the use and execution of this protocol, please refer to Lee et al.¹.

Measuring the electrophysiological responses of olfactory receptor neurons (ORNs) to odorants in spotted lanternfly (SLF) (Lycorma delicatula) is crucial for understanding how this invasive sap-feeding planthopper locates host plants, aggregates, and mates. Here, we present a protocol for single sensillum recording from SLF labial ORNs to measure their sensitivity, specificity, and response dynamics to odorants. We describe the steps for preparing odorant cartridges, mounting the labium, setting up the electrophysiology rig, recording neuronal responses to odorants, and analyzing data. For complete details on the use and execution of this protocol, please refer to Dweck and Claire.¹.

The innate and adaptive immune systems, though often studied separately, interact deeply and respond to stimuli simultaneously, with leukocytes displaying a range of pro- to anti-inflammatory phenotypes. This protocol details a procedure for characterizing murine innate and adaptive immune phenotypes using a 40-color full-spectral flow cytometry panel. We describe steps for organ collection, sample preparation, immunofluorescent staining, and acquisition to reproducibly and cost-effectively study tissue-resident leukocytes, their subpopulations, and inflammatory status in various organs.

Delivering microRNA (miRNA) to treat cardiac disease is a significant challenge, and selecting an efficient delivery method to target the affected organ is critical for therapeutic success. Here, we present a protocol for delivering miRNA to the heart of rats with heart failure using adeno-associated virus (AAV9) coupled with a hydrodynamic transfusion strategy. We describe steps for inducing heart failure, echocardiography, and AAV9 application. Further, we provide a comprehensive procedure to detect miRNA expression in cardiac tissues using stem-loop RT-qPCR. For complete details on the use and execution of this protocol, please refer to Mushtaq et al.¹.

Biotin ligase-based proximity ligation is a widely used, highly effective technique for the study of in vivo protein-protein interactions. However, there are few reports and little consensus on the most effective methods for studying the proximal interactomes of secreted factors. Here, we present a protocol for studying extracellular proximal interactomes using an adaptation of TurboID/BioID2-based proximity ligation. We describe steps for cell preparation, sample collection, and initial processing. We then detail procedures for biotinylated protein enrichment, on-bead digestion, and post-pull-down processing. For complete details on the use and execution of this protocol, please refer to Peeney et al.¹.

The use of 3D cancer models to study therapy response has a great advantage over conventional 2D cell culture. Here, we present an optimized protocol for breast cancer spheroid and rod-shaped microtissue formation using MicroTissues mold systems. We describe steps for cast formation and treating the 3D models with DNA-damaging agents. We then detail procedures for analyzing the 3D models by whole-mount immunostaining and confocal imaging of fixed samples or with the use of live-cell reporters.

Continuing advancements in human pluripotent stem cell (hPSC)-derived complex three-dimensional (3D) cardiac tissues require the development of novel technologies or adaptation of existing technologies to understand the physiology of the derived 3D cardiac tissues. In this protocol, we describe the use of multielectrode array (MEA) and sharp electrode electrophysiology techniques to investigate the electrical properties of 3D cardiac organoids. This protocol deciphers the electrical behavior of 3D cardiac organoids at both the single-cell level and tissue level.

The use of archival formalin-fixed paraffin-embedded (FFPE) tissue samples for biochemical analyses is problematic because of the formation of a Schiff base, leading to low protein and metabolite yields during analytical extractions. Here, we overcome this issue using a unified protocol on FFPE tissue for metabolomics and proteomics analyses. Using 20 mg of wet mass tissue, this protocol consistently extracted more than 50 metabolites (across 11 classes of metabolites) and over 900 proteins.

Reticulocyte isolation from peripheral blood is crucial for hematological research. Here, we present a protocol for high-quality reticulocyte enrichment from small blood quantities obtained from alpha-thalassemic and healthy participants. We describe steps for Ficoll and Percoll gradient centrifugation to obtain a reticulocyte-enriched fraction, followed by negative immunomagnetic separation to remove granulocytes and platelets. This technique allows enriched reticulocyte isolation for multi-omics hematological analysis. Additionally, we detail procedures to recover peripheral blood mononuclear cells (PBMCs) and erythrocytes from the original blood sample.