STAR Protocols最新文献

Protein synthesis could control spermatogenic cell fate transitions. Here, we present a protocol for visualization and quantification of newly synthesized proteins by click-chemistry-based immunofluorescence within specific spermatogenic cell types in the mice testicular lumen. We detail the processes for O-propargyl-puromycin (OPP) incorporation, antibody incubation, confocal microscope imaging, and subsequent quantification methods. This protocol is not limited to spermatogenic cells and can be adapted to investigate protein synthesis in other testicular cell types and various tissue-specific cell populations. For complete details on the use and execution of this protocol, please refer to Zou et al.¹.

The mechanisms of DNA double-strand break (DSB) production and repair vary throughout the cell cycle. Here, we provide a protocol to quantify DSB production and repair in G1, S, and G2 phases of asynchronous adherent cells by coupling the staining of DSBs and cell-cycle markers with automated high-content fluorescence microscopy. We describe steps for cell seeding, treatment, staining, imaging, and analysis. This protocol is broadly applicable for monitoring DSB dynamics at single-cell level throughout the cell cycle. For complete details on the use and execution of this protocol, please refer to Geraud et al.¹.

In the exact nuclear Overhauser enhancement (eNOE) method, tight upper and lower interatomic distance restraints for structure calculations and comparison are extracted from NMR nuclear Overhauser enhancement spectroscopy (NOESY) spectra of biomolecules recorded with different mixing times. Here, we present a protocol for exporting the resonance assignments of NOESY spectra from CCPNmr (v.2) into nmrDraw to obtain peak intensities. We then detail procedures for converting them into distances using either the CYANA- or MATLAB-based "exact NOE by relaxation matrix analysis" (eNORA) program.

Innovations in electrophysiological recordings and computational analytic techniques enable high-resolution analysis of neural traveling waves. Here, we present a protocol for the detection and analysis of traveling waves from multi-day microelectrode array human electrophysiological recordings through a multi-linear regression statistical approach using point estimator data. We describe steps for traveling wave detection, feature characterization, and propagation pattern analysis. This protocol may improve our understanding of the coordination of neurons during non-oscillatory neural dynamics. For complete details on the use and execution of this protocol, please refer to Smith et al.¹.

Extracting the choroid plexus (ChP) for histology provides a comprehensive view of the whole tissue and is crucial for understanding its structure and cellular interactions. Here, we present a protocol for dissecting out the lateral, third, and fourth ventricle ChP from mice and the immunostaining process. We also detail procedures for preparing whole-mount ChP tissue and imaging techniques, such as tile scan and Z-stacks. For complete details on the use and execution of this protocol, please refer to Xu et al.¹.

Large-scale production of induced pluripotent stem cell (iPSC)-derived neurons is valuable in disease modeling and drug discovery. Here, we describe a workflow to engineer a doxycycline-inducible NGN2 (neurogenin 2) cassette into the AAVS1 (adeno-associated virus integration site 1) locus and differentiate positive clones into neurons. iPSCs are electroporated with ribonucleoprotein and a donor plasmid. The positive clone rate is maximized with homology-directed repair enhancement, antibiotic selection, and fluorescence. Validated clones are differentiated into neurons in 5 days at a scale of billions. These neurons can be cryopreserved or maintained for months. For complete details on the use and execution of this protocol, please refer to Shan et al.¹.

The rapid growth of the aviation industry has significantly impacted global climate change. We present a protocol to assess the climate effects of global aviation emissions. We describe steps for calculating annual emissions; greenhouse gas concentrations; effective radiative forcing from gases, contrails, and aerosols; and temperature variations under different scenarios. We also outline how to analyze the potential impacts of mitigation strategies. For complete details on the use and execution of this protocol, please refer to Cui et al.¹.

Recombinant microtubule (MT)-severing proteins are valuable for studying their mechanisms of action; however, purifying them in an active state is challenging. Here, we provide a protocol to obtain biologically active and highly pure recombinant GFP-UNC-45A, a novel ATP-independent MT-severing protein, from E. coli. We describe steps for using GST-affinity and spin columns and detail procedures for assessing the activity of GFP-UNC-45A with in vitro MTs along with GFP-katanin as a positive control. The purified proteins can be used for downstream applications to study their functions. For complete details on the use and execution of this protocol, please refer to Habicht et al.¹.

Pairwise similarity mapping (PaSiMap), implemented in Jalview, serves as an alternative to principal component analysis (PCA) for analyzing protein sequence relationships without requiring pre-computed multiple sequence alignments. It excels at distinguishing between systematic and random differences in datasets. Here, we present a protocol for sequence clustering with PaSiMap in Jalview. We describe steps for installation, sequence importing, and running PaSiMap analyses. We also detail procedures for plotting and data interpretation in RStudio, offering a streamlined approach for visualizing and analyzing protein sequence relationships effectively.