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Lipid particles found in circulating extracellular fluids such as blood or lymph are essential for cellular homeostasis, metabolism and survival. Such particles provide essential lipids and fats which enable cells to synthesize new membranes and regulate different biochemical pathways. Imbalance in lipid particle metabolism can cause pathological states such as atherosclerosis. Here, elevated low-density lipoprotein (LDL) accumulation leads to fat-filled lesions or plaques in arterial walls. In this chapter, we provide a detailed set of protocols for the rapid and safe purification of lipid particles from human blood using high-speed ultracentrifugation. We provide a detailed set of assays for further analysis of the biochemical and cellular properties of these lipid particles. By combining these assays, we can better understand the complex roles of different lipid particles in normal physiology and disease pathology., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The endothelial response to vascular endothelial growth factor A (VEGF-A) regulates many aspects of animal physiology in health and disease. Such VEGF-A-regulated phenomena include vasculogenesis, angiogenesis, tumor growth and progression. VEGF-A binding to receptor tyrosine kinases such as vascular endothelial growth factor receptor 2 (VEGFR2 ) activates multiple signal transduction pathways and changes in homeostasis, metabolism, gene expression, cell proliferation, migration, and survival. One such VEGF-A-regulated response is a rapid rise in cytosolic calcium ion levels which modulates different biochemical events and impacts on endothelial-specific responses. Here, we present a series of detailed and robust protocols for evaluating ligand-stimulated cytosolic calcium ion flux in endothelial cells. By monitoring an endogenous endothelial transcription factor (NFATc2 ) which displays calcium-sensitive redistribution, we can assess the relevance of cytosolic calcium to protein function. This protocol can be easily applied to both adherent and non-adherent cultured cells to evaluate calcium ion flux in response to exogenous stimuli such as VEGF-A., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

A major obstacle to studying membrane proteins by biophysical techniques is the difficulty in producing sufficient amounts of materials for functional and structural studies. To overexpress the target membrane protein heterologously, especially an eukaryotic protein, a key step is to find the optimal host expression system and perform subsequent expression optimization. In this chapter, we describe protocols for screening membrane protein production using bacterial and insect cells, solubilization screening, large-scale production, and commonly used affinity chromatography purification methods. We discuss general optimization conditions, such as promoters and tags, and describe current techniques that can be used in any laboratory without specialized expensive equipment. Especially for insect cells, GFP fusions are particularly useful for localization and in-gel fluorescence detection of the proteins on SDS-PAGE. We give detailed protocols that can be used to screen the best expression and purification conditions for membrane protein study.

The perineuronal net (PNN) is a specialized extracellular matrix structure that surrounds subpopulations of neurons in the central nervous system (CNS). The appearance of PNNs on the cell surface marks the closure of the critical period during development and has been observed to reduce synaptic plasticity. Perineuronal nets comprise hyaluronan, chondroitin sulfate proteoglycans (CSPGs), link proteins, tenascin-R, and other components, some of which are substrates for a disintegrin-like and metalloprotease domain with thrombospondin type 1 motifs (ADAMTS) proteases. There is a high heterogeneity of PNNs in the CNS. Depending on which part of the CNS is studied, the PNNs may be observed surrounding the soma, or both the soma and proximal dendrites. The most robust marker for PNN is a lectin called Wisteria floribunda agglutinin. Here, we describe a method for preparing tissue for visualization of PNNs in CNS.

Single-molecule localization microscopy allows the ability to image fluorescence labeled molecular targets at nanoscale resolution. However, for many biological questions the ability to provide tissue and cellular context in addition to these high resolution data is eminently informative. Here, we describe a procedure to achieve this aim by correlatively imaging human cardiac tissue first at the nanoscale with direct stochastic optical reconstruction microscopy (dSTORM) and then at the diffraction limit with conventional confocal microscopy.

Angiogenesis is an important determinant of tissue function, from delivery of oxygen and other substrates to removal of waste products, in health and disease (e.g., adaptive or pathological remodelling). The phenotype and functional responses of endothelial cells are conditioned by systemic humoral signals and local environmental factors, including the haemodynamic forces that act upon them. Here we describe some interventions that have been helpful in unraveling the integrative nature of the complex in vivo response, and quantitative assessment of angiogenesis in muscle.

The patch clamp technique revolutionized the study of ion channels and is considered the gold standard of measuring ion channel activity, from the academic laboratory to industrial-scale drug screening. This technique enables the study of ion channels, from single molecules up to the whole-cell ion channel population, and in their native environment. Whilst the study of single protein molecular behavior is the ultimate goal of biophysicists from all fields, this is a routine ability for ion channel specialists. This chapter is aimed at helping the beginner to design patch clamp experiments and to obtain the fundamental micropipette configurations with mammalian cells: cell-attached patch, whole cell, inside-out patch, and outside-out patch.

Cysteine contains a highly reactive thiol group and therefore under oxidizing conditions a disulfide bond can form between a pair of cysteines that are juxtaposed in the close vicinity, which can be only reversed by reducing agents. These attributes have been elegantly exploited to study the functional role of an interaction or contact between two adjacent domains that are present in ion channels or virtually in any proteins, by introducing double cysteine substitutions at the domain interface and measuring changes in the ion channel functions arising from cross-linking the two substituted cysteines via formation of a disulfide bond. Here I describe this cysteine-based cross-linking approach.

Perforated whole-cell patch-clamp is a variant of the patch-clamp technique used to measure the sum activity of ion channels in the plasma membrane of a single cell. Its defining feature is that electrical access to the cell is obtained through inclusion of a pore-forming antibiotic in the patch pipette which perforates the sealed patch of membrane in contact with the patch pipette. The antibiotic pores allow equilibration of small monovalent ions between the patch pipette and the cytosol whilst maintaining endogenous levels of divalent ions such as Ca(2+) and signalling molecules such as cAMP. Therefore, the perforated patch-clamp technique is ideal for studying ion channels whilst maintaining the integrity of second messenger signalling cascades. Other benefits of using perforated patch-clamp over conventional patch-clamp include reduced current rundown and stable whole-cell recording lasting >1 h. In this chapter, the application of the perforated patch-clamp technique for the study of heterologously expressed Kv7 potassium channels will be discussed in detail including benefits and limitations of the technique.

Individual types of ion channels and other membrane proteins are typically expressed only at low levels in their native membranes, rendering their isolation by conventional purification techniques difficult. The heterologous over-expression of such proteins is therefore usually a prerequisite for their purification in amounts suitable for structural and for many functional investigations. The most straightforward expression host, suitable for prokaryote membrane proteins and some proteins from eukaryotes, is the bacterium Escherichia coli. Here we describe the use of this expression system for production of functionally active polytopic membrane proteins and methods for their purification by affinity chromatography in amounts up to tens of milligrams.

This chapter describes immunochemistry-based methods to investigate recycling of membrane proteins at the cell surface. Two methods are described, one qualitative and the other quantitative. Both methods consist of two rounds of extracellular antibody capture. Firstly, a primary antibody is captured by an extracellular epitope presented by the target membrane protein and is subsequently internalized. Secondly, the primary antibody-labelled protein is recycled back to the membrane where it is captured by a probe--conjugated secondary antibody. In the qualitative assay, the probe is a fluorophore, which can be imaged by fluorescence microscopy. In the quantitative assay, the probe is horse-radish peroxidase (HRP) and enzyme activity can be assayed by chemiluminescence.

Nucleofection is a transfection method used to introduce substrates such as cDNA plasmids into primary cells or other cell lines. The method can be successfully applied to cells that are considered difficult to transfect or suffer from low transfection efficiency as seen with traditional transfection techniques. Neurons in primary cultures retain many properties of their in vivo state and therefore, in many instances, are considered better experimental systems than immortalized cell lines, thus becoming increasingly desirable cell types for biomedical research. However, being post-mitotic, primary neuronal cultures are particularly difficult to transfect using routine transfection reagents. There is therefore a growing need for the efficient delivery of expression vectors into such neuronal cultures. In this chapter we will discuss the application of nucleofection for the heterologous expression of genes in primary neuronal cultures. We also discuss the advantage of this technique relative to other conventional methods, and describe a reliable method for transfection of cultured rat dorsal root ganglion (DRG) and trigeminal (TG) neurons.

Electrophysiological recordings from an acutely sliced preparation provide information on ionic currents and excitability of native neurons under near physiological conditions. Although this technique is commonly used on central nervous system structures such as spinal cord and brain, structures within the peripheral nervous system (including sensory ganglia and fibers) have proven to be much more difficult to study in acute preparations. Here we describe a method for patch-clamp recordings from rat dorsal root ganglion (DRG) slices.

Exogenous expression of genes in mammalian neurons represents a substantial experimental challenge because of the low efficiency of commercially available liposomal transfection reagents for nondividing cells and considerable toxicity of viral transfection systems. In this chapter, we discuss application of the "biolistic" particle delivery system for heterologous expression of genes in primary neuron cultures. The method is based on the direct introduction of cDNA of interest into the nucleus by penetration with DNA-coated gold particles. With this approach, cDNA expression is independent of cell cycling and proliferation and is similar to intranuclear microinjection, with both avoiding cDNA delivery through the cytosol. Examples of successful transfection using PDS of rat superior cervical ganglion and trigeminal ganglion neurons are discussed.