Protocol for building synthetic protocell membranes that sense redox using synthetic phospholipids and natural lipids.

Sensing is a critical function of artificial cells; however, this is challenging to realize using bottom-up approaches. Here, we present a protocol for building protocell membranes that sense cues important for redox biochemistry and signaling by combining synthetic phospholipids and natural lipids. We detail procedures for building giant unilamellar vesicles as protocell models that fluoresce in response to the biologically significant redox agents peroxynitrite, hydrogen peroxide, and hydrogen sulfide. For complete details on the use and execution of this protocol, please refer to (i) Gutierrez and Aggarwal et al.1 as well as (ii) Erguven and Wang et al.2.

Protocol to generate, purify, and analyze antibody-oligonucleotide conjugates from off-the-shelf antibodies.

Antibody-oligonucleotide conjugates (AOCs) are a fast-expanding modality for targeted delivery of therapeutic oligonucleotides to tissues. Here, we present a protocol to generate, purify, and analyze AOCs from off-the-shelf antibodies. We describe steps to conjugate single/double-stranded oligonucleotides bearing amine handles to linkers and, then, to antibodies using well-established chemistry. In addition, we provide details regarding the purification techniques and analytical methods suitable for AOC. This protocol can be applied for several purposes where AOC is a modality of interest. For complete details on the use and execution of this protocol, please refer to Rady et al.1.

Protocol for detecting nitrative stress in biological lipid membranes in murine cells and tissues.

Detection of nitrative stress is crucial to understanding redox signaling and pathophysiology. Dysregulated nitrative stress, which generates high levels of peroxynitrite, can damage lipid membranes and cause activation of proinflammatory pathways associated with pulmonary complications. Here, we present a protocol for implementing a peroxynitrite-sensing phospholipid to investigate nitrative stress in murine cells and lung tissue. We detail procedures for sensing ONOO- in stimulated cells, both ex vivo and in vivo, using murine models of acute lung injury (ALI). For complete details on the use and execution of this protocol, please refer to Gutierrez and Aggarwal et al.1.

Protocol for matching protein localization to synapse morphology in primary rat neurons by correlative super-resolution microscopy.

Super-resolution imaging provides unprecedented visualization of sub-cellular structures, but the two main techniques used, single-molecule localization microscopy (SMLM) and stimulated emission depletion (STED), are not easily reconciled. We present a protocol to super-impose nanoscale protein distribution reconstructed with SMLM to sub-cellular morphology obtained in STED. We describe steps for tracking cells on etched coverslips and registering images from two different microscopes with 30-nm accuracy. In this protocol, synaptic proteins are mapped in the dendritic spines of primary neurons. For complete details on the use and execution of this protocol, please refer to Inavalli et al.1.

Protocol for transcriptome-wide mapping of small-molecule RNA-binding sites in live cells.

Small molecules targeting RNA can be valuable chemical probes and potential therapeutics. The interactions between small molecules, particularly fragments, and RNA, however, can be difficult to detect due to their modest affinities and short residence times. Here, we present a protocol for mapping the molecular fingerprints of small molecules in vitro and throughout the human transcriptome in live cells. We describe steps for compound treatment, cross-linking, RNA extraction, fragmentation, and pull-down. We then detail procedures for RNA sequencing and data analysis. For complete details on the use and execution of this protocol, please refer to Tong et al.1.

Intracellular cholesterol visualization in brain tissue using D4H∗.

Studying cholesterol biology in the brain has been greatly hindered by the lack of adequate cholesterol visualization techniques. Here, we present a protocol for using a high-affinity cholesterol probe D4H∗-mCherry as a histology reagent in mouse or human brain tissue. We describe steps for D4H∗ tissue treatment and crosslinking leading to stable labeling of intracellular membrane cholesterol. Furthermore, co-labeling with Rab5 endosomal marker and optimized buffers to reduce background enable punctate cholesterol visualization within the organelle membranes.

Protocol for screening α-synuclein PET tracer candidates in vitro and ex vivo.

Alpha-synuclein (α-Syn) positron emission tomography (PET) imaging is a valuable approach for diagnosing and monitoring synucleinopathies-related diseases, such as Parkinson disease. Here, we present a protocol for screening potential α-Syn PET tracers using in vitro and ex vivo approaches. We describe steps for employing recombinant pre-formed fibrils and conducting screening procedures on neuronal models, mouse models, and patients' brain tissue sections to assess the specificity and selectivity of the candidate compounds. For complete details on the use and execution of this protocol, please refer to Xiang et al. (2023).1.

Protocol for producing phosphoramidate using phosphorus fluoride exchange click chemistry.

Phosphorus fluoride exchange (PFEx) is a catalytic click reaction that involves exchanging high oxidation state P-F bonds with alcohol and amine nucleophiles, reliably yielding P-O- and P-N-linked compounds. Here, we describe steps for preparing a phosphoramidic difluoride and performing two sequential PFEx reactions to yield a phosphoramidate through careful catalyst selection. We then detail procedures for handling and quenching potentially toxic P-F-containing compounds to ensure user safety when conducting PFEx reactions. For complete details on the use and execution of this protocol, please refer to Sun et al.1.

Protocol for organelle-specific cysteine capture and quantification of cysteine oxidation state.

Pinpointing functional, structural, and redox-sensitive cysteines is a central challenge of chemoproteomics. Here, we present a protocol comprising two dual-enrichment cysteine chemoproteomic techniques that enable capture of cysteines (Cys-LoC) and quantification of cysteine oxidation state (Cys-LOx) in a localization-specific manner. We describe steps for utilizing TurboID-mediated protein biotinylation for enrichment of compartment-specific proteins, followed by click-mediated biotinylation and enrichment of cysteine-containing peptides. Thus, changes to compartment-specific cysteine identification and redox state can be assessed in a variety of contexts. For complete details on the use and execution of this protocol, please refer to Yan et al. (2023).1.

Protocol for measuring protein synthesis in specific cell types in the mouse brain using in vivo non-canonical amino acid tagging.

The fluorescent non-canonical amino acid tagging (FUNCAT) technique has been used to visualize newly synthesized proteins in cell lines and tissues. Here, we present a protocol for measuring protein synthesis in specific cell types in the mouse brain using in vivo FUNCAT. We describe steps for metabolically labeling newly synthesized proteins with azidohomoalanine, which introduces an azide group into the polypeptide. We then detail procedures for binding a fluorophore-conjugated alkyne to the azide group to allow its visualization. For complete details on the use and execution of this protocol, please refer to tom Dieck et al. (2012)1 and Hooshmandi et al. (2023).2.

Electrophoretic mobility shift assays (EMSAs) for in vitro detection of protein-nucleic acid interactions.

Protein-nucleic acid interactions drive some of the most important physiological events in cells. Here, we present a protocol for detecting protein-DNA or protein-RNA interactions in vitro. We describe steps for labeling nucleic acid species and electrophoretic mobility shift assays (EMSAs). This protocol can be used to confirm suspected in vivo interactions using recombinantly expressed/purified proteins of interest and a nucleic acid substrate. It can further be used to investigate mutations that can disrupt interaction or compensatory mutations that restore it. For complete details on the use and execution of this protocol, please refer to Mansouri-Noori et al.1.

Protocol for preparation of electrifiable carbon membrane reactor for non-oxidative alkane dehydrogenation.

A membrane reactor (MR) offers a solution to overcome thermodynamic equilibrium limitations by enabling in situ product separation, enhancing product yields and energy efficiency. Here we present a protocol for synthesizing a carbon MR that couples a H2-permeable carbon molecular sieve hollow fiber membrane and a metal supported on zeolite catalyst for non-oxidative propane and ethane dehydrogenation. We describe steps for catalyst preparation, membrane fabrication, and MR construction. The as-developed MR has significant improvements in alkene yield and a record-high stability. For complete details on the use and execution of this protocol, please refer to Liu et al.1.

Protocol for quantifying LC3B FRET biosensor activity in living cells using a broad-to-sensitive data analysis pipeline.

Here, we present a protocol to comprehensively quantify autophagy initiation using the readout of the microtubule associated protein 1 light chain 3 beta (LC3B) Förster's resonance energy transfer (FRET) biosensor. We describe steps for cell seeding, transfection, FRET/FLIM (fluorescence lifetime imaging microscopy) imaging, and image analysis. This protocol can be useful in any physiology- or disease-related paradigm where the LC3B biosensor can be expressed to determine whether autophagy has been initiated or is stalled. The analysis pipeline presented here can be applied to any other genetically encoded FRET sensor imaged using FRET/FLIM. For complete details on the use and execution of this protocol, please refer to Gökerküçük et al.1.

Application of bioluminescence resonance energy transfer assays in primary mouse neuronal cultures.

Bioluminescence resonance energy transfer (BRET) is widely employed for real-time monitoring of G protein-coupled receptor activity, interactions, and trafficking in heterologous cell lines, yet its use in neuronal systems remains limited. Here, we present a protocol to apply BRET assays to primary neuronal cultures from mouse embryos. We describe steps and key concepts for generating plasmid constructs and lentivirus preparations, plating and lentiviral transduction of primary cultured neurons in 96-well plates, and BRET data collection and analysis. For complete details on the use and execution of this protocol, please refer to George et al.1.

Protocol for quantum dot-based cell counting and immunostaining of pulmonary arterial cells from patients with pulmonary arterial hypertension.

Currently, there is no protocol for growing and culturing primary pulmonary arterial cells (PACs) available from the Pulmonary Hypertension Breakthrough Initiative (PHBI). Here, we present a protocol for cultivating and maintaining three major PACs collected from patients with pulmonary arterial hypertension (PAH): endothelial (PAH-ECs), smooth muscle (PAH-SMCs), and adventitial cells (PAH-ADCs). We describe steps for obtaining PACs from PHBI, evaluating the growth of cells labeled with quantum dots (QDs), and staining endothelial cell (EC) markers for immunofluorescence imaging. For complete details on the use and execution of this protocol, please refer to Al-Hilal et al.1.

Click-chemistry-based protocol for detecting 4-octyl-itaconate-alkylated proteins in primary mouse macrophages.

4-Octyl itaconate (4-OI), a derivative of itaconate, inhibits inflammation by alkylating its target proteins. Here, we present a click-chemistry-based protocol for detecting 4-OI-alkylated proteins in mouse primary bone-marrow-derived macrophages (BMDMs) by using an itaconate-alkyne (ITalk) probe. We describe steps for culturing and treating BMDMs and details on using click chemistry in the cell lysate. We also detail procedures for detecting alkylated proteins by western blot. For complete details on the use and execution of this protocol, please refer to Su et al.1.

Protocol for preparing formalin-fixed paraffin-embedded musculoskeletal tissue samples from mice for spatial transcriptomics.

Here, we present a protocol for using spatial transcriptomics in bone and multi-tissue musculoskeletal formalin-fixed paraffin-embedded (FFPE) samples from mice. We describe steps for tissue harvesting, sample preparation, paraffin embedding, and FFPE sample selection. We detail procedures for sectioning and placement on spatial slides prior to imaging, decrosslinking, library preparation, and final analyses of the sequencing data. The complete protocol takes ca. 18 days for mouse femora with adjacent muscle; of this time, >50% is required for mineralized tissue decalcification. For complete details on the use and execution of this protocol, please refer to Wehrle et al.1 and Mathavan et al.2.

Protocol to identify S-acylated proteins in hippocampal neurons using ω-alkynyl fatty acid analogs and click chemistry.

S-acylation, commonly palmitoylation, is the addition of fatty acids to cysteines to regulate protein localization and function. S-acylation detection has been hampered by limited sensitivity and selectivity in low-protein, costly samples like cultured neurons. Here, we present a protocol for sensitive and selective bioorthogonal labeling and click-chemistry-based detection of S-acylated proteins in primary hippocampal neurons. We describe steps for metabolically labeling neurons with alkynyl fatty acid, click chemistry, NeutrAvidin-based capture, and elution with hydroxylamine.

Protocol for the comprehensive biochemical and cellular profiling of small-molecule degraders using CoraFluor TR-FRET technology.

Comprehensive characterization of small-molecule degraders, including binary and ternary complex formation and degradation efficiency, is critical for bifunctional ligand development and understanding structure-activity relationships. Here, we present a protocol for the biochemical and cellular profiling of small-molecule degraders based on CoraFluor time-resolved fluorescence resonance energy transfer (TR-FRET) technology. We describe steps for labeling antibodies and proteins, tracer saturation binding, binary target engagement, ternary complex profiling, and off-rate determination. We then detail procedures for the quantification of endogenous and GFP fusion proteins in cell lysates. For complete details on the use and execution of this protocol, please refer to Ichikawa et al.1.

Protocol for preparation and staining of chromosomes isolated from mouse and human tissues for conventional and molecular cytogenetic analysis.

The study of chromosomes without or with molecular DNA probes provides crucial insight for understanding research findings, as well as refining diagnosis, prognosis, and therapeutics in clinical settings. Here, we present a protocol for chromosome preparation, conventional G-banding, locus-specific fluorescent in situ hybridization, and spectral karyotyping for both mouse and human samples. This protocol optimizes the preparation of chromosomes from mouse and human cells for subsequent conventional and molecular cytogenetic analysis. For complete details on the use and execution of this protocol, please refer to Binz et al.1.