STAR Protocols最新文献

R2 retrotransposons can be harnessed to insert genes at targeted sites by all-RNA delivery, presenting a new technology for next-generation biotherapeutics. Here, we report a protocol for evaluating the gene integration activity of R2 retrotransposons in mammalian cells. We describe the construction of vectors separately expressing R2 protein and donor, the process of liposome transfection, and flow cytometry. This protocol provides a useful reporter system, which can be applied to evaluate the activity of other new retrotransposon systems. For complete details on the use and execution of this protocol, please refer to Chen et al.¹.

Measuring the cytotoxicity of human T cells is a valuable but challenging task. We present a protocol for assessing the ability of polyclonal human T cells to kill target cells upon T cell receptor (TCR) triggering. We describe steps for preparing L and T cells for the assay and incubation of target cells with effector cells. We detail procedures for assessing cell death by flow cytometry. This protocol can be applied to small numbers of effector cells isolated from patient samples. For complete details on the use and execution of this protocol, please refer to Wang et al.¹.

Here, we present a protocol to alter the production of alternatively spliced mRNA variants, without affecting the overall gene expression, through CRISPR-Cas9-engineered genomic mutations in mice. We describe steps for designing guide RNA to direct Cas9 endonuclease to consensus splice sites, producing transgenic mice through pronuclear injection, and screening for desired mutations in cultured mammalian cells using a minigene splicing reporter. Splice isoform-specific mouse mutants provide valuable tools for genetic analyses beyond loss-of-function and transgenic alleles. For complete details on the use and execution of this protocol, please refer to Dailey-Krempel et al.¹ and Johnson et al.².

Strand-optimized Smart-seq (So-Smart-seq) can capture a comprehensive transcriptome from low-input samples. This technique detects both polyadenylated and non-polyadenylated RNAs, inclusive of repetitive RNAs, while excluding highly abundant ribosomal RNAs. So-Smart-seq preserves strand information and minimizes 5' to 3' coverage bias. We describe steps for the analysis of single mouse preimplantation embryos, including embryo isolation, library preparation, ribosomal cDNA depletion, and initial data processing. The protocol may be adapted for other low-input samples and the detection of small RNAs of <200 nt. For complete details on the use and execution of this protocol, please refer to Wei et al.¹.

Neutral lipids affect the immunosuppressive function of myeloid-derived suppressor cells (MDSCs). Here, we present a protocol for measuring neutral lipids in MDSCs using BODIPY from mouse mammary tumor derived from triple-negative breast cancer cells, 4T1, which is applicable to other mammary tumors of interest. We describe steps for 4T1 cell culture, single-cell isolation from tumors, staining of cells with antibodies and BODIPY, and flow cytometry. Furthermore, we introduce alternative protocols with MDSC sorting to overcome risk of cell death by BODIPY. For complete details on the use and execution of this protocol, please refer to Kim et al.¹.

Post-translational modifications (PTMs) of histone H4 play significant roles in the regulation of chromatin status. Here, we present a protocol for semisynthesis of histone H4 by sortase-mediated ligation (SML). We describe steps for solid-phase peptide synthesis of H4R40C(1-42), recombinant expression and purification of H4(41-102), expression and purification of eSrt(2A-9), and preparation of acrylamidine. We then detail procedures for SML of histone H4. This protocol can also be applied to the preparation of homogenous proteins with PTMs. For complete details on the use and execution of this protocol, please refer to Xiao et al.^1,2.

As light sheet fluorescence microscopy (LSFM) becomes widely available, reconstruction of time-lapse imaging will further our understanding of complex biological processes at cellular resolution. Here, we present a comprehensive workflow for in toto capture, processing, and analysis of multi-view LSFM experiments using the ex vivo mouse embryo as a model system of development. Our protocol describes imaging on a commercial LSFM instrument followed by computational analysis in discrete segments, using open-source software. Quantification of migration and morphodynamics is included. For complete details on the use and execution of this protocol, please refer to Dominguez et al.¹.

Chimeric antigen receptors (CARs) are synthetic molecules composed of an extracellular antigen-binding domain and an intracellular signaling domain, leading to tonic signaling and manufacturing challenges. We present a protocol for the expansion of tonic CARs by using a Food and Drug Administration (FDA)-approved kinase inhibitor, dasatinib. We report steps for T cell transduction with retrovirus, expansion and verification of CAR quality using flow cytometry, and killing assay. At only 30 nM, dasatinib improves tonic CAR T cell proliferation and quality after expansion. For complete details on the use and execution of this protocol, please refer to Caulier et al.¹.

Plant vacuoles are essential organelles that respond to developmental and environmental signals. Here, we present a protocol for staining the vacuole lumen in Arabidopsis root cells, enabling precise visualization of vacuolar dynamics. We describe steps for preparing plant material and staining with commonly used fluorescent dyes. We then detail procedures for visualizing vacuoles in the blue, green, and red emission spectra, allowing for their combined use with a variety of compatible fluorescent-tagged protein markers. For complete details on the use and execution of this protocol, please refer to Dubrovsky et al.,¹ Fricker,² Bassil et al.,³ Grzam et al.,⁴ and Stefano et al.⁵.

Sodium influx induced by hepatitis B virus (HBV) enhances activation of the NLRP3 inflammasome, in synergy with potassium efflux signals in macrophages. Here, we present a protocol for conducting patch-clamp assays to measure HBV-mediated sodium influx in THP-1 macrophages. We describe steps for differentiating, resting, and activating THP-1 macrophages and then outline the procedures for whole-cell patch-clamp recordings. For complete details on the use and execution of this protocol, please refer to Wang et al.¹.