Protocol for live neuron imaging analysis of basal surface fraction and dynamic availability of the dopamine transporter using DAT-pHluorin.

Surface availability of the dopamine (DA) transporter (DAT) critically influences DA transmission. Here, we present a protocol that describes the preparation of mouse ventral midbrain neurons, the expression of a new optical sensor, DAT-pHluorin, and the utilization of this sensor to analyze the surface availability of DAT in live neurons via fluorescent microscopy. This approach allows quantitative measures of basal surface DAT fraction under genetic backgrounds of interest and live trafficking of DAT in response to psychoactive substances. For complete details on the use and execution of this protocol, please refer to Saenz et al.1.

Protocol for modeling acquired resistance to targeted therapeutics in adherent and suspension cancer cell lines via in situ resistance assay.

Acquired resistance to oncogene-targeted therapies is the major driver of mortality for patients with cancer. Here, we present a 6-to-16-week assay to model the development of acquired resistance in adherent and suspension cancer cell lines. We describe steps for determining therapeutic dose, assaying acquired resistance, and testing combination therapies. This protocol is a high-throughput, cost-effective, and scalable method to model acquired drug resistance to established and newly developed therapies. For complete details on the use and execution of this protocol, please refer to Sealover et al.1 and Theard et al.2.

Protocol for CRISPR-Cas9 genome editing of a swine cell line via electroporation.

Genome editing technology is being used in animals for a variety of purposes, including improvement of animal and public health outcomes. Characterization of genome editing reagents and anticipated genomic alterations is an essential step toward the development of an edited animal. Here, we present a protocol for genome editing in the swine testicular (ST) cell line. We describe steps for evaluating CRISPR-Cas9 complex functionality in vitro, delivering editing molecules into cells by transfection, and assessing target editing via Sanger sequencing.

Antioxidant Properties of Postbiotics: An Overview on the Analysis and Evaluation Methods.

Antioxidants found naturally in foods have a significant effect on preventing several human diseases. However, the use of synthetic antioxidants in studies has raised concerns about their potential link to liver disease and cancer. The findings show that postbiotics have the potential to act as a suitable alternative to chemical antioxidants in the food and pharmaceutical sectors. Postbiotics are bioactive compounds generated by probiotic bacteria as they ferment prebiotic fibers in the gut. These compounds can also be produced from a variety of substrates, including non-prebiotic carbohydrates such as starches and sugars, as well as proteins and organic acids, all of which probiotics utilize during the fermentation process. These are known for their antioxidant, antibacterial, anti-inflammatory, and anti-cancer properties that help improve human health. Various methodologies have been suggested to assess the antioxidant characteristics of postbiotics. While there are several techniques to evaluate the antioxidant properties of foods and their bioactive compounds, the absence of a convenient and uncomplicated method is remarkable. However, cell-based assays have become increasingly important as an intermediate method that bridges the gap between chemical experiments and in vivo research due to the limitations of in vitro and in vivo assays. This review highlights the necessity of transitioning towards more biologically relevant cell-based assays to effectively evaluate the antioxidant activity of postbiotics. These experiments are crucial for assessing the biological efficacy of dietary antioxidants. This review focuses on the latest applications of the Caco-2 cell line in the assessment of cellular antioxidant activity (CAA) and bioavailability. Understanding the impact of processing processes on the biological properties of postbiotic antioxidants can facilitate the development of new food and pharmaceutical products.

Protocol to investigate G protein-coupled receptor signaling kinetics and concentration-dependent responses using ONE-GO biosensors.

ONE vector G protein optical (ONE-GO) biosensors are versatile tools to measure the activity of G protein-coupled receptors (GPCRs) in cells. The availability of ONE-GO biosensors for ten active Gα subunits representative of all four G protein families (Gs, Gi/o, Gq/11, and G12/13) permits the study of virtually any GPCR. Here, we present a protocol to implement ONE-GO biosensors in cell lines to investigate GPCR signaling kinetics and concentration-dependent responses. We describe steps for cell culture and transfection, response measurement, and data analysis. For complete details on the use and execution of this protocol, please refer to Janicot et al.1.

Protocol for detecting endogenous GPCR activity in primary cell cultures using ONE-GO biosensors.

ONE vector G protein Optical (ONE-GO) biosensors can measure the activity of endogenously expressed G protein-coupled receptors (GPCRs) in primary cells. By detecting G proteins that belong to all four families (Gs, Gi/o, Gq/11, G12/13) across cell types, these biosensors provide high experimental versatility. We first describe steps to express ONE-GO biosensors in primary cells using lentiviral transduction. We then detail how to carry out measurements and subsequent analysis to quantify changes in bioluminescence resonance energy transfer (BRET) reporting on endogenous GPCR activity. For complete details on the use and execution of this protocol, please refer to Janicot et al.1.

Protocol for detecting homologous recombination activity in cancer cells by adenovirus-based functional assay.

Cancer cells with homologous recombination deficiency (HRD) exhibit a distinctive vulnerability to poly(ADP-ribose) polymerase inhibitors (PARPis). To address the limitations of existing methodologies incapable of providing real-time insights into homologous recombination (HR) status, we present an adenovirus-based functional assay designed to quantify cellular HR activity. Here, we delineate the step-by-step procedure for producing the adenovirus harboring an HR reporter, processing primary cells, and assessing HR activity in primary ovarian cancer cells. For complete details on the use and execution of this protocol, please refer to Lee et al.1.

Protocol to calculate the synergy of drug combinations in organoids and primary cells from murine tumors.

Evaluating the synergy of drug combinations is crucial in advancing treatment regimens. Here, we present a protocol to establish primary cells and organoids from murine tumors and calculate drug synergy. We describe all necessary cell culture procedures, including establishing primary cultures, setting up treatment groups, and detecting cell viability. We then outline how to calculate the synergy score based on a bioinformatical pipeline. This approach applies to any disease model in which a combination of drugs needs to be evaluated. For complete details on the use and execution of this protocol, please refer to Ku et al.1.

Protocol for oil red O staining of low-density lipoproteins for in vivo cell treatment.

Low-density lipoproteins (LDLs) are the most abundant circulating lipoproteins and the most critical factor in the development of atherosclerosis. This protocol allows the staining of LDLs with oil red O to monitor particle uptake in bright-field microscopy. Here, we describe how to stain isolated LDLs using oil red O and how to use them to monitor LDL uptake in time-lapse experiments or fixed cells.

Protocol to identify E3 ligases amenable to biodegraders using a cell-based screening.

Here, we provide a protocol for the identification of E3 ubiquitin ligases that are functional when implemented as biodegraders using a cell-based screening assay. We describe steps for establishing a stable cell line expressing a GFP-tagged protein of interest (POI), preparing a sub-library of E3 ligases to screen, and performing the cell-based screening. This protocol can be broadly applied to identify any functional E3 ligase in a biodegrader setting. For complete details on the use and execution of this protocol, please refer to Cornebois et al.1.

Protocol for lipid mediator profiling and phenotyping of human M1- and M2-monocyte-derived macrophages during host-pathogen interactions.

Here, we present a protocol for primary, human immune cell isolation and stimulation for lipid mediator profiling. We describe steps for the isolation of monocytes from human leukocyte concentrates via density centrifugation and differentiation/polarization toward M1- or M2-monocyte-derived macrophages (MDMs). We detail stimulation approaches of MDMs with live bacteria or influenza A virus for lipid mediator profiling and sample preparation for subsequent analysis, such as enzyme expression, mRNA analysis, or surface marker determination. For complete details on the use and execution of this protocol, please refer to Jordan et al.1.

Protocol to measure and analyze protein interactions in mammalian cells using bioluminescence resonance energy transfer.

Bioluminescence resonance energy transfer (BRET) allows to quantitate protein interactions in intact cells. Here, we present a protocol for measuring BRET due to transient interactions of oncogenic K-RasG12V in plasma membrane nanoclusters of HEK293-EBNA cells. We describe steps for seeding, transfecting, and replating cells. We then detail procedures for their preparation for BRET measurements on a CLARIOstar microplate reader and detailed data analysis. For complete details on the use and execution of this protocol, please refer to Steffen et al.1.

Protocol to identify receptors of secreted proteins through CRISPR-Cas9 whole-genome screening technology.

The interaction between cell surface receptors and their ligands is crucial for intercellular communication. However, current techniques for identifying direct receptor-ligand interactions remain limited. Here, we present a protocol to identify receptors of secreted proteins using a genome-scale CRISPR-Cas9 knockout genetic screening approach. We describe steps for creating a single-guide RNA (sgRNA) lentivirus library, infecting stable Cas9-MCF7 cells, staining with tagged Cholesin, and sorting non-binding cells via flow cytometry. We then detail procedures for extracting DNA, amplifying sgRNAs, and sequencing. For complete details on the use and execution of this protocol, please refer to Hu et al.1.

Protocol for in vitro transcribing mRNAs with defined poly(A)-tail lengths and visualizing sequential PABP binding.

Quantifying the number of proteins that interact with mRNAs, in particular with poly(A) tails of mRNAs, is crucial for understanding gene regulation. Biochemical assays offer significant advantages for this purpose. Here, we present a protocol for synthesizing mRNAs with accurate, length-specific poly(A) tails through a PCR-based approach. We also describe steps for an in vitro (i.e., cell-free) approach for visualizing the sequential binding of Cytoplasmic Poly(A)-Binding Proteins (PABPCs) to these poly(A) tails. We detail quality control steps throughout the procedure. For complete details on the use and execution of this protocol, please refer to Grandi et al.1.

An Inducible Luminescent System to Explore Parkinson's Disease-Associated Genes.

With emerging genetic association studies, new genes and pathways are revealed as causative factors in the development of Parkinson's disease (PD). However, many of these PD genes are poorly characterized in terms of their function, subcellular localization, and interaction with other components in cellular pathways. This represents a major obstacle towards a better understanding of the molecular causes of PD, with deeper molecular studies often hindered by a lack of high-quality, validated antibodies for detecting the corresponding proteins of interest. In this study, we leveraged the nanoluciferase-derived LgBiT-HiBiT system by generating a cohort of tagged PD genes in both induced pluripotent stem cells (iPSCs) and iPSC-derived neuronal cells. To promote luminescence signals within cells, a master iPSC line was generated, in which LgBiT expression is under the control of a doxycycline-inducible promoter. LgBiT could bind to HiBiT when present either alone or when tagged onto different PD-associated proteins encoded by the genes GBA1, GPNMB, LRRK2, PINK1, PRKN, SNCA, VPS13C, and VPS35. Several HiBiT-tagged proteins could already generate luminescence in iPSCs in response to the doxycycline induction of LgBiT, with the enzyme glucosylceramidase beta 1 (GCase), encoded by GBA1, being one such example. Moreover, the GCase chaperone ambroxol elicited an increase in the luminescence signal in HiBiT-tagged GBA1 cells, correlating with an increase in the levels of GCase in dopaminergic cells. Taken together, we have developed and validated a Doxycycline-inducible luminescence system to serve as a sensitive assay for the quantification, localization, and activity of HiBiT-tagged PD-associated proteins with reliable sensitivity and efficiency.

Protocol to study the direct binding of proteins to RNA:DNA hybrids or RNA-DNA chimeras in living cells using cross-linking immunoprecipitation.

RNA-binding proteins (RBPs) are involved in many biological processes. The direct interaction between protein and RNA can be studied using cross-linking immunoprecipitation (CLIP) techniques in living cells. Here, we present a protocol to characterize the direct binding of proteins to RNA:DNA hybrids or RNA-DNA chimeras in living cells using CLIP. We describe steps for RNA-protein UV-C cross-linking in living cells, isolating RNA-protein complexes, RNA labeling, and extracting nucleic acid. We then detail procedures for nuclease treatment and nucleic acid migration.

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.

Gz Enhanced Signal Transduction assaY (GZESTY) for GPCR deorphanization.

G protein-coupled receptors (GPCRs) are key pharmacological targets, yet many remain underutilized due to unknown activation mechanisms and ligands. Orphan GPCRs, lacking identified natural ligands, are a high priority for research, as identifying their ligands will aid in understanding their functions and potential as drug targets. Most GPCRs, including orphans, couple to Gi/o/z family members, however current assays to detect their activation are limited, hindering ligand identification efforts. We introduce GZESTY, a highly sensitive, cell-based assay developed in an easily deliverable format designed to study the pharmacology of Gi/o/z-coupled GPCRs and assist in deorphanization. We optimized assay conditions and developed an all-in-one vector employing novel cloning methods to ensure the correct expression ratio of GZESTY components. GZESTY successfully assessed activation of a library of ligand-activated GPCRs, detecting both full and partial agonism, as well as responses from endogenous GPCRs. Notably, with GZESTY we established the presence of endogenous ligands for GPR176 and GPR37 in brain extracts, validating its use in deorphanization efforts. This assay enhances the ability to find ligands for orphan GPCRs, expanding the toolkit for GPCR pharmacologists.

Protocol to induce neurodegeneration in a local area of the mouse brain by stereotaxic injection.

In vivo models of brain pathology are crucial for studying neurological diseases. Here, we present a protocol to induce a pathological condition in a mouse brain area by local injection of neurotoxic stimulus. We describe steps for preparing reagents, stereotaxic injection procedures to induce neurodegeneration in the hippocampus, and preparation of brain sections to examine the induced model. This protocol is useful for studying how local pathology affects other brain areas and neighbor cells and its functional consequences in behavior. For complete details on the use and execution of this protocol, please refer to Zhang 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.