STAR Protocols最新文献

Killer yeast produce antimicrobial proteins, often together with a self-protective immunity factor. Here, we present a protocol for analyzing the toxic, immune, and suicidal phenotypes in Saccharomyces cerevisiae. We describe steps for assessing toxicity via halo assays, suicidality through spot assays, and immunity using microtiter plate growth assays. We detail procedures for yeast culturing, performing the assays, and data analysis. For complete details on the use and execution of this protocol, please refer to Prins et al.¹.

Protein import into the mitochondria is required for organellar function. Inefficient import can result in the stalling of mitochondrial precursors inside the translocase of the outer membrane (TOM) and blockage of the mitochondrial entry gate. Here, we present a protocol to assess the clogging of TOM by mitochondrial precursors in human cell lines. We describe how the localization of mitochondrial precursors can be determined by cellular fractionation. We then show how co-immunoprecipitation can be used to test the stalling of precursors inside TOM. For complete details on the use and execution of this protocol, please refer to Kim et al.¹.

Extended pluripotent stem cells (EPSCs) possess a high differentiation capacity, potentially as a superior seed resource for generating cardiomyocytes. Here, we present a protocol for generating feeder-free EPSCs (ffEPSCs), cardiomyocytes, and engineered heart tissues (EHTs). We describe steps for converting human embryonic stem cells or induced pluripotent stem cells (ESCs/iPSCs) into ffEPSCs, followed by their long-term maintenance, cryopreservation, seed preservation, and differentiation into cardiomyocytes. We then detail procedures for constructing and culturing three-dimensional EHTs followed by their contraction force measurement and optical mapping. For complete details on the use and execution of the protocol, please refer to Zheng et al.¹ and Li et al.².

Chromatin immunoprecipitation (ChIP) is used to investigate genome binding by transcription factors, but it can be problematic. We present a protocol to isolate fixed DNA-protein complexes from mouse liver prior to chromatin shearing. We describe steps for liver disaggregation and cross-linking, DNA-protein complex isolation, chromatin shearing, and quality control analysis as well as procedures for ChIP, DNA purification, and ChIP analysis. This protocol yields high-quality samples using commercial antibodies. For complete details on the use and execution of this protocol, please refer to Akl et al.¹.

Visualizing the expression of mRNAs using traditional in situ hybridization is often hampered by obstacles including weak signal, high background, and poor probe specificity. Here, we present a protocol utilizing RNAscope (ACD) to overcome these obstacles and detect multiple types of mRNAs simultaneously in whole-mount adult Drosophila brains. We further describe how mRNAs can be reliably quantified in any cells that can be targeted by common binary expression systems such as Gal4/UAS and labeled by immunohistochemistry. For complete details on the use and execution of this protocol, please refer to De et al.¹.

Here, we present a protocol for the rapid functional screening of gene editing and addition strategies in patient-derived organoids using the deep-learning-based tool DETECTOR (detection of targeted editing of cystic fibrosis transmembrane conductance regulator [CFTR] in organoids). We describe steps for wet-lab experiments, image acquisition, and CFTR function analysis by DETECTOR. We also detail procedures for applying pre-trained models and training custom models on new customized datasets. For complete details on the use and execution of this protocol, refer to Bulcaen et al.¹.

The superior mesenteric artery (SMA), originating from the abdominal aorta, provides blood supply for the spleen, pancreas, intestine, and part of the colon. Here, we present a protocol for studying the SMA in the same mouse by combining non-invasive micro-ultrasound imaging and SMA dissection. We then detail procedures for tissue preparation, sectioning, and histological analysis. By allowing accurate identification of the SMA by ultrasound and anatomical dissection, this protocol can provide insights into SMA-related diseases in animal models.

Drug resistance has emerged as a critical challenge in clinical cancer treatment. Here, we present a high-throughput screening protocol to identify therapeutic small-molecule inhibitors against drug-resistant cancer cells. We detail the steps for constructing drug-resistant cell models, executing the chemical screening process, and performing data analysis and validation. This protocol facilitates the rapid identification of therapeutic strategies for different types of drug-resistant cancers and aids in studying mechanisms. For complete details on the use and execution of this protocol, please refer to Zhang et al.¹.

Antigen array increases B cell receptor (BCR) triggering and the titer of antibodies elicited by subunit vaccines. Here, we present a protocol for multivalent antigen display by synthetic virosomes: preformed liposomes bearing glycoprotein spike proteins from enveloped viruses. We describe how to customize lipid stoichiometry within preformed liposomes and attach user-defined antigens via covalent and/or non-covalent interactions. In addition to generating vaccine research tools, this protocol demonstrates how two-dimensional membrane array resolves and activates exceptionally weak but critical virus-receptor interactions.

Human induced pluripotent stem cell (iPSC)-derived oligodendrocytes are a powerful tool for studying aberrant myelination in neurodegenerative and neurodevelopmental disorders; however, they often fail to myelinate in vitro. Here, we present a protocol for axonal ensheathment and perinodal segmentation using an ex vivo model. We describe steps for preparing Shiverer mouse brain slice cultures, oligodendrocyte transplantation, visualization, and analysis. This approach suits multiple culture formats, highlighting the potential for the screening of myelin-modulating drugs and compounds in a cost- and time-effective manner, while reducing animal use.