STAR Protocols最新文献

Defects in retinal metabolism have been linked to the onset and progression of various retinal diseases. Herein, we provide a protocol for measuring bioenergetics in dissociated mouse retinal photoreceptors. We outline detailed instructions for obtaining morphologically intact and viable photoreceptor cells from adult mice and preparing the cells for metabolic analysis using a SeahorseXFe24 analyzer. This protocol allows a real-time assessment of mitochondrial respiration and glycolysis in retinal photoreceptors in response to genetic modifications or pathological insults in mouse models.

Three-dimensional (3D) and porous scaffolds made from nanocellulosic materials hold significant potential in tissue engineering (TE). Here, we present a protocol for fabricating self-standing (nano)cellulose-based 3D scaffolds designed for in vitro testing of cells from skin and cartilage tissues. We describe steps for preparation of nanocellulose ink, scaffold formation using 3D printing, and freeze-drying. We then detail post-processing procedures to enhance mechanical properties, stability, and biocompatibility. This protocol offers researchers a framework for developing versatile and sustainable biomaterials for regenerative medicine. For complete details on the use and execution of this protocol, please refer to Mohan et al.¹ and Štiglic et al.².

Fecal microbiota transplantation (FMT) is clinically applied, while oral FMT (oral fecal gavage [OFG]) is preferred for experimental mice. Here, we present a protocol for OFG in antibiotic-pretreated mice, demonstrating the progressive, time-dependent evolution of the gut microbiota in the recipients. We describe steps for fecal sample collection and preparation procedures, oral gavage, and monitoring gut microbiota changes. This protocol serves as a general guide for reshaping the gut microbiota in recipient mice for various experimental applications. For complete details on the use and execution of this protocol, please refer to Yang et al.¹.

Here, we present a protocol to generate dual-target compounds (DT-CPDs) interacting with two distinct target proteins using a transformer-based chemical language model. We describe steps for installing software, preparing data, and pre-training the model on pairs of single-target compounds (ST-CPDs), which bind to an individual protein, and DT-CPDs. We then detail procedures for assembling ST- and corresponding DT-CPD data for specific protein pairs and evaluating the model's performance on hold-out test sets. For complete details on the use and execution of this protocol, please refer to Srinivasan and Bajorath.¹.

Preparing high-titer virus and performing accurate titer determination are critical to subsequent experiments. However, not all applied recombinant rabies viruses, such as the L-deleted virus,¹ are equipped with fluorescent proteins for titration by fluorescence-activated cell sorting (FACS). Here, we present a quantitative reverse-transcription PCR (RT-qPCR) approach for titrating recombinant rabies virus. We describe steps for preparing standards for RT-qPCR, rabies virus genome RNA extraction, and reverse transcription of virus RNA. We then detail procedures for RT-qPCR for titration and stereotaxic rabies virus injection for titer verification.

Contrast transfer function (CTF) estimation is essential to the data processing workflow of cryo-electron tomography (cryoET). Here, we present a protocol for CTF estimation of the cryoET tilt series with CTFMeasure. CTFMeasure can estimate the CTF parameters together with the absolute tilt angle offset of the sample. We describe steps for configuring the input files and estimating the CTF parameters and the absolute tilt angle offset of the input tilt series. We then detail procedures for visualizing the power spectra and analyzing the output files. For complete details on the use and execution of this protocol, please refer to Zhang et al.¹.

Gut-microbiome-combined metabolomics studies in cerebrovascular disease highlight the microbiota-gut-brain axis in neurological disorders. Here, we present a protocol for correlating the gut microbiome and metabolomics in patients with intracranial aneurysms. We describe steps for sample collection, fecal genomic DNA extraction, rRNA PCR amplification, sequencing library construction, and rRNA sequencing. We then detail procedures for metabolite extraction, liquid chromatography-tandem mass spectrometry (LC-MS/MS) non-targeted metabolomics sequencing, and ELISA for cerebrospinal fluid and plasma samples. Finally, we perform combined multi-omics analysis. For complete details on the use and execution of this protocol, please refer to Xu et al.¹.

Cell competition is a quality control mechanism that promotes elimination of suboptimal cells relative to fitter neighbors. Cancer cells exploit these mechanisms for expansion, but the underlying molecular pathways remain elusive. Here, we present a protocol for generating matrix-free microtissues recapitulating cellular interactions between intestinal cancer and hepatocyte-like cells using microscopy or transcriptomics/proteomics. We describe steps for generating and differentiating liver progenitor organoids and microtissue formation. We then detail procedures for immunofluorescence staining, mounting microtissues, and sorting cells. For complete details on the use and execution of this protocol, please refer to Krotenberg Garcia et al.¹.

Due to their small size and transparency, larval zebrafish are a useful model for whole-brain imaging. Here, we present a protocol for the visualization of phosphorylated Rps6, a marker of mechanistic target of rapamycin complex 1 (mTORC1) activity, in the zebrafish brains at 5 days post fertilization (dpf), using whole-mount immunofluorescence and light-sheet microscopy. We describe steps for sample preparation, storage, staining, and imaging. This protocol can also be modified for staining with antibodies against other proteins. For complete details on the use and execution of this protocol, please refer to Doszyn et al.¹.

Obtaining viable cell suspension that accurately represents the diversity of complex tissues is challenging due to the distinct characteristics of each cell type. Here, we present a protocol for preparing a single-cell suspension of the zebrafish embryonic whole heart, detailing steps for heart extraction, cell dissociation, quantification, and quality assessment. This suspension is compatible with downstream analysis on various single-cell platforms. For details on the use and execution of this protocol, please refer to Abu Nahia et al.¹.