microRNAs (miRNAs) are noncoding RNAs of approximately 24 nucleotides long that are expressed in multiple tissues and organs. miRNAs regulate the expression of specific target genes, and their expression profile can prov...microRNAs (miRNAs) are noncoding RNAs of approximately 24 nucleotides long that are expressed in multiple tissues and organs. miRNAs regulate the expression of specific target genes, and their expression profile can provide insight into the cell biology altered by diseases or drug treatments. Research has demonstrated that several miRNAs regulate muscle differentiation and the specification of fiber type. Hence, an established tool to routinely quantify miRNA abundance is of the utmost importance. The stem-loop quantitative reverse transcription PCR (RT-qPCR) is a methodology that allows the analysis of a specific miRNA with high sensitivity. Upon hybridization with a structured stem-loop oligonucleotide to the 3' end of the target miRNA, a retro-transcription is possible for further qPCR quantification. In this chapter, we describe a protocol to quantify miRNAs in dissected mouse fast and slow-twitch muscle.
Neonatal rodent cardiomyocytes serve as a valuable in vitro model for investigating the cellular and molecular mechanisms of the heart, offering an alternative to the technical challenges associated with isolating and ma...Neonatal rodent cardiomyocytes serve as a valuable in vitro model for investigating the cellular and molecular mechanisms of the heart, offering an alternative to the technical challenges associated with isolating and maintaining adult cardiomyocytes. Importantly, studying atrial and ventricular cardiomyocytes separately is crucial due to their distinct functional and genetic properties. Here, we present a protocol for the simultaneous isolation and culture of neonatal mouse atrial and ventricular cardiomyocytes. This procedure includes the dissection of neonatal mouse hearts at postnatal day 0 to day 2(P0-P2), separation of atrial appendages and ventricles, and sequential enzymatic digestion using trypsin and collagenase. The chamber-specific identities of isolated cardiomyocytes were validated through gene expression analyses and immunofluorescence staining with atrial and ventricular markers. The resulting cardiomyocyte populations exhibit high viability and retain chamber-specific characteristics, rendering them suitable for a wide array of experimental applications under both physiological and pathological conditions. Potential applications include physiological assessments (e.g., calcium transient analysis and patch clamp), morphological studies, biochemical assays, and drug screening.
Cardiomyocyte maturation represents the final stage of functional development in the heart. During this critical process, fetal cardiomyocytes gradually acquire adult phenotypes and become capable of sustaining billions...Cardiomyocyte maturation represents the final stage of functional development in the heart. During this critical process, fetal cardiomyocytes gradually acquire adult phenotypes and become capable of sustaining billions of contraction-relaxation cycles throughout an organism's lifespan. This transition is driven by gene expression changes that remodel cardiomyocyte structure, metabolism, and functions. In mice, cardiomyocyte maturation takes place during embryonic day (E)18.5 to postnatal two weeks, during which the heart loses its regenerative capacity in response to injury. Hence, characterizing the functional and molecular changes during mouse cardiomyocyte maturation and how this process is regulated provides invaluable insights into heart development and disease. Cardiomyocyte-specific analysis requires isolating cardiomyocytes from other cardiac cell populations. Here, we present a standardized protocol for consistent isolation of neonatal and postnatal mouse cardiomyocytes. Leveraging enzymatic and mechanical digestion, coupled with non-cardiomyocyte depletion via pre-plating, our protocol enables high-quality cardiomyocyte isolation in approximately three hours. In addition, we detail methods for cardiomyocyte culture, molecular, and functional characterization.
Mouse cardiac fibroblasts (FBs) have been widely used as in vitro models for studying fundamental biological processes and mechanisms underlying cardiac pathologies, as well as identifying potential therapeutic targets....Mouse cardiac fibroblasts (FBs) have been widely used as in vitro models for studying fundamental biological processes and mechanisms underlying cardiac pathologies, as well as identifying potential therapeutic targets. Cardiac FBs preparations are relatively easy to culture in a dish and can be manipulated using molecular and pharmacological tools. Because FBs rapidly decrease cell cycle division and proliferative rate after birth, they are prone to undergo phenotypic changes and senescence in cell culture as soon as a few passages. Therefore, primary cultures of differentiated FBs with amplified cell cycle from embryonic animals are required for accurate and reliable experiments. Below, we will describe a method that provides good cell yield, quality, and viability of mouse embryonic cardiac fibroblasts (MEFs) primary cultures from E16 CD-1 mice.
Eye development is a complex process that begins during the first stages of embryonic development; during its formation, a series of cell proliferation, migration, and differentiation events occur. A frequently used labo...Eye development is a complex process that begins during the first stages of embryonic development; during its formation, a series of cell proliferation, migration, and differentiation events occur. A frequently used laboratory method is tissue staining with two dyes known as hematoxylin-eosin (H&E). H&E staining allows us to observe the arrangement of the cell layers and the formation of characteristic structures. It is fundamental for research in developmental biology since it facilitates a comprehensive analysis of the formation of organs and systems.In this chapter, we will describe the procedure for acquiring H&E-stained images of the embryonic eye structure, specifically focusing on the retina. This technique serves as a fundamental tool for understanding ocular morphogenesis, organogenesis, and structural modifications resulting from experimental interventions.
Enteric neural crest cells (ENCCs) are progenitor cells in the developing gut that give rise to the enteric nervous system (ENS). Mechanisms that regulate the migration, proliferation, and differentiation of these cells...Enteric neural crest cells (ENCCs) are progenitor cells in the developing gut that give rise to the enteric nervous system (ENS). Mechanisms that regulate the migration, proliferation, and differentiation of these cells are crucial to the proper morphogenesis of the intestinal neural network during embryogenesis. Despite the relatively low population of these cells within the gut tissue, methods for isolation and in vitro culture have been established to enrich this cell population and enable investigations into molecular pathways that govern ENS development. Here, we describe a method to generate ENCC-derived neurospheres from the mouse embryonic gut at embryonic day (E) 13.5 by neural selective media enrichment.
Migration of enteric neural crest cells (ENCCs) within the developing embryonic gut is key to the formation of the enteric nervous system (ENS). Investigating factors that guide the migration of these cells along the gut...Migration of enteric neural crest cells (ENCCs) within the developing embryonic gut is key to the formation of the enteric nervous system (ENS). Investigating factors that guide the migration of these cells along the gut tube could aid in understanding the pathogenesis and developing potential therapy for ENS developmental defects, such as those present in Hirschsprung disease. However, limitations exist on appropriate study models to allow the live tracing of these migrating cells within the developing intestine. This protocol describes a mouse embryonic gut explant model utilizing a Sox10-Venus transgenic mouse to fluorescently label migrating ENCCs within the gut for live-cell imaging during culturing.
Lung development is a complex process that requires the input of various signaling pathways to coordinate the specification and differentiation of multiple cell types. Ex vivo culture of the lung is a highly useful techn...Lung development is a complex process that requires the input of various signaling pathways to coordinate the specification and differentiation of multiple cell types. Ex vivo culture of the lung is a highly useful technique that represents an attractive model for investigating various processes critical to lung development, function, and disease pathology. Ex vivo cultured lungs remain comparable to the in vivo lung both in structure and function, which makes them more suitable than cell cultures for physiological studies. Lung explant cultures offer several significant advantages for studying morphogenic events that guide lung development, including budding, branching, and fusion. Additionally, these cultures preserve the native physiological interactions between cells in the developing lung, enabling investigations into the direct and indirect signalling taking place between tissues and cells throughout the developmental process. Studying the temporal and spatial control of gene expression by transcriptional factors using different reporters to understand their regulatory function at different moments of development is another valuable advantage of lung explant culture.
Accurate visualization of embryonic vasculature provides critical insights into cardiovascular development and the origins of congenital heart defects. Here, we describe a protocol for a low-viscosity plastic resin injec...Accurate visualization of embryonic vasculature provides critical insights into cardiovascular development and the origins of congenital heart defects. Here, we describe a protocol for a low-viscosity plastic resin injection into the fetal mouse heart, which then polymerizes to create stable casts of the great vessels. These casts preserve fine vascular architecture and enable the detection of structural abnormalities, such as aortic arch artery anomalies and ventricular septal defects. This method provides a robust and reproducible approach for investigating both normal and pathological cardiovascular networks in rodent embryos.
Traditional histological sectioning has been widely used to examine congenital heart defects (CHDs) in mouse models, providing detailed insights into tissue and cellular architecture. However, this approach is time-consu...Traditional histological sectioning has been widely used to examine congenital heart defects (CHDs) in mouse models, providing detailed insights into tissue and cellular architecture. However, this approach is time-consuming, labor-intensive, and can miss subtle defects due to the critical need for precise serial sections. In contrast, three-dimensional imaging of cleared embryonic hearts using light sheet fluorescence microscopy offers a more efficient and comprehensive solution. This technique enables the rapid acquisition of z-stacks, including thousands of sections, without manual sectioning, which can be reconstructed into high-resolution 3D models. As a result, it significantly improves the detection and visualization of CHDs in mouse fetuses.
In situ hybridization (ISH) is a powerful method for analyzing gene expression patterns in tissue sections and whole-mount specimens. Three-dimensional (3D) imaging of ISH signals helps to understand the gene expression...In situ hybridization (ISH) is a powerful method for analyzing gene expression patterns in tissue sections and whole-mount specimens. Three-dimensional (3D) imaging of ISH signals helps to understand the gene expression patterns in the complex structure of developing organs. This chapter describes the protocol for creating 3D images of ISH signals in a cost-effective and time-efficient manner using the correlative microscopy and block-face imaging (CoMBI) system. A unique feature of the CoMBI system is the ability to obtain both serial block-face images and sections as needed for preferential staining, such as hematoxylin and eosin staining. Correlating section images and 3D data helps to annotate gene expression patterns and understand the anatomy of developing organs. We describe the protocol for the sample preparation (ISH and embedding), acquisition of serial block-face images, and image processing to create 3D images.
The heart is a dynamic organ that undergoes rapid morphological and mechanical changes during early embryonic development. Characterizing these early moments is important for our understanding of proper embryonic develop...The heart is a dynamic organ that undergoes rapid morphological and mechanical changes during early embryonic development. Characterizing these early moments is important for our understanding of proper embryonic development and the treatment of heart disease. Traditionally, tomographic imaging modalities and fluorescence-based microscopy are excellent approaches for visualizing structural features and gene expression patterns, respectively, and connecting aberrant gene programs to pathological phenotypes. However, these approaches typically require static samples or fluorescent markers, which can limit the amount of information we can derive from the dynamic and mechanical changes that regulate heart development. Optical coherence tomography (OCT) is unique in this context because it enables the acquisition of three-dimensional structural images and four-dimensional (3D + time) functional imaging of living mouse embryos without the need for fixation or contrast agents. In this chapter, we focus on how OCT can visualize heart morphology at different stages of development and provide cardiodynamic information to reveal the mechanical properties of the developing heart.
Genotyping is a valuable tool for identifying organisms that carry genetic variations, including insertions and deletions. This method involves extracting DNA from animal tissue samples and subsequently amplifying genomi...Genotyping is a valuable tool for identifying organisms that carry genetic variations, including insertions and deletions. This method involves extracting DNA from animal tissue samples and subsequently amplifying genomic regions of interest using the polymerase chain reaction (PCR). The amplified product is analyzed by agarose gel electrophoresis based on DNA size.
G protein-coupled receptors (GPCRs) are vital cellular signaling machinery that are dependent on the membrane environment in which they reside. For instance, cholesterol has been reported to be critical for different asp...G protein-coupled receptors (GPCRs) are vital cellular signaling machinery that are dependent on the membrane environment in which they reside. For instance, cholesterol has been reported to be critical for different aspects of GPCR function, such as ligand binding, G-protein coupling, receptor stability, organization, and dynamics. Multiscale simulation approaches have helped complement experimental approaches in probing the interplay of GPCRs with their surrounding lipids. The large complexity and diversity of membrane lipids, coupled with their stochastic interactions spanning multiple timescales, necessitate the use of such a multi-pronged approach. In this chapter, we discuss the considerations needed to set up atomistic and coarse-grain simulations, and the related analysis methods to extract robust and meaningful values of receptor-lipid interactions. We discuss crucial aspects of the simulation setup, including selecting appropriate lipid compositions. We then explore both qualitative and quantitative approaches for analyzing GPCR-lipid interactions, ranging from occupancy values and enrichment indices to free energy calculations and residence time analyses. In addition, we highlight approaches to identify synergistic co-binding, as well as competitive binding of lipids to GPCRs, a phenomenon that is often neglected in model membrane studies. This comprehensive overview will help researchers to perform robust and informative simulations to elucidate the intricate interplay between GPCRs and their surrounding lipid bilayer.
Microfluidic diffusional sizing (MDS) is a powerful technique in bioanalytical research, offering a broad spectrum of applications based on its simple yet effective principle-relating diffusion speed to particle size. Ho...Microfluidic diffusional sizing (MDS) is a powerful technique in bioanalytical research, offering a broad spectrum of applications based on its simple yet effective principle-relating diffusion speed to particle size. However, its reliance on selectively fluorescently labeled analytes poses challenges when studying lipid nanoparticles or complex biological samples, such as crude cell extracts.Fluorescent universal lipid labeling for MDS (FULL-MDS) is a sparse, noncovalent labeling method designed to overcome this limitation. Using widely available fluorophores such as Nile blue and Nile red, FULL-MDS enables robust hydrodynamic size determination of lipid nanoparticles in both simple synthetic and complex biological samples. Moreover, its labeling approach is straightforward, cost-effective, and compatible with other hydrodynamic sizing techniques, making it highly adaptable for diverse research applications. FULL-MDS therefore holds significant potential for advancing the characterization of lipid nanoparticles in both fundamental and applied sciences.
Biophysical characterization of membrane protein folding and stability provides a direct molecular correlation of folding and stability with protein function. Here, we describe how mitochondrial outer membrane β-barrels...Biophysical characterization of membrane protein folding and stability provides a direct molecular correlation of folding and stability with protein function. Here, we describe how mitochondrial outer membrane β-barrels can be overproduced, and how structures of folding intermediates and per-residue thermodynamic stability are measured using intrinsic tryptophan fluorescence in near-native membranes.
G protein-coupled receptors (GPCRs) are cellular nanomachines that act as signaling hubs and account for ~40% of current drug targets. Endocytosis of GPCRs represents a major regulatory mechanism to sustain their downstr...G protein-coupled receptors (GPCRs) are cellular nanomachines that act as signaling hubs and account for ~40% of current drug targets. Endocytosis of GPCRs represents a major regulatory mechanism to sustain their downstream signaling within a stringent spatiotemporal regime. This is achieved by internalizing a fraction of the plasma membrane receptor population, thereby reducing the available pool of receptors for signaling at the cell membrane. In this article, we describe a detailed step-by-step hands-on guide that we have successfully used to monitor GPCR endocytosis and intracellular trafficking using serotonin receptor as a representative GPCR. For this, we carried out a judicious combination of flow cytometry and quantitative colocalization of confocal microscopic images to explore receptor endocytosis and trafficking. Notably, we applied this approach to demonstrate the role of membrane lipids (such as cholesterol) on endocytosis and trafficking of the serotonin receptor. Finally, we highlight important points and caveats to be considered, while carrying out endocytosis and trafficking experiments.
Many structural characterization techniques either require a solid sample or for the sample to be placed on a substrate. This can be a challenge when the structure depends on the concentration of environmental parameters...Many structural characterization techniques either require a solid sample or for the sample to be placed on a substrate. This can be a challenge when the structure depends on the concentration of environmental parameters such as pH and salt. Small-angle X-ray/neutron scattering (SAXS/SANS) is an excellent tool that can be used for in situ, non-invasive, structural characterization of soft matter and biological systems in solution. In this chapter, we present how to prepare lipid vesicles as a relevant model system for cell membranes and their interactions with surface-active agents. We include a brief introduction of scattering theory and the basics of SAXS/SANS, how to analyze and interpret the data, as well as discussing some complementary techniques that can elucidate different aspects/properties of your sample.
Brewster angle microscopy (BAM) is a highly surface-sensitive, noninvasive technique used to study the reflectivity and lateral domain structures of Langmuir monolayers at the air-water interface. This experimental syste...Brewster angle microscopy (BAM) is a highly surface-sensitive, noninvasive technique used to study the reflectivity and lateral domain structures of Langmuir monolayers at the air-water interface. This experimental system uniquely allows for the control of lateral compression while simultaneously measuring surface pressure and visualization of domains. The primary advantage of BAM over other methods is its ability to directly measure in situ the intrinsic properties of samples without requiring chemical modifications, external probes or further manipulations like transfer to a solid support. This chapter outlines the basic theory of BAM and introduces a new method for determining the refractive index of the monolayer, a key parameter often missing in data analysis. By combining the refractive index with reflectivity measurements, it becomes possible to calculate the thickness of films as a function of lateral packing in lipid, protein, and lipid/protein films.
Nanodiscs provide a useful approach for experimental studies focused on elucidating the structure and function of membrane proteins. The self-assembly process offers an efficient way for optimizing the composition and st...Nanodiscs provide a useful approach for experimental studies focused on elucidating the structure and function of membrane proteins. The self-assembly process offers an efficient way for optimizing the composition and stoichiometry of the lipid mixture, scaffold protein, and the target membrane protein, resulting in soluble lipoprotein particles with a highly homogeneous size distribution. As a result, this system is now commonly used for many biophysical and biochemical applications, resulting in hundreds of new publications every year. In this review, we give an overview of some of the recent results that describe various aspects of lipid-protein interactions being investigated using the Nanodisc system, focusing on the novel methods and presenting some of the advantages of this methodology.