Cell Biology

Expansion microscopy is an innovative method that enables super-resolution imaging of biological materials using a simple confocal microscope. The principle of this method relies on the physical isotropic expansion of a biological specimen cross-linked to a swellable polymer, stained with antibodies, and imaged. Since its first development, several improved versions of expansion microscopy and adaptations for different types of samples have been produced. Here, we show the application of ultrastructure expansion microscopy (U-ExM) to investigate the 3D organization of the green algae Chlamydomonas reinhardtii cellular ultrastructure, with a particular emphasis on the different types of sample fixation that can be used, as well as compatible staining procedures including membranes.

Graphical overview

Neurons communicate via synapses—specialized structures that consist of a presynaptic terminal of one neuron and a postsynaptic terminal of another. As knowledge is emerging that mutations in molecules that regulate synaptic function underpin many neurological disorders, it is crucial to elucidate the molecular mechanisms regulating synaptic function to understand synaptic strength, plasticity, modulation, and pathology, which ultimately impact neuronal circuit output and behavior. The presynaptic calyx of Held is a large glutamatergic presynaptic terminal in the auditory brainstem, which due to its accessibility and the possibility to selectively perform molecular perturbations on it, is an ideal model to study the role of presynaptic proteins in regulating synaptic function. In this protocol, we describe the use of confocal imaging and three-dimensional reconstruction of the calyx of Held to assess alterations in gross morphology following molecular perturbation. Using viral-vector delivery to perform molecular perturbations at distinct developmental time points, we provide a fast and cost-effective method to investigate how presynaptic proteins regulate gross morphology such as surface area and synapse volume throughout the lifetime of a neuronal circuit.

Key features

• Confocal imaging and 3D reconstruction of presynaptic terminals.

• Used with a virus-mediated expression of mEGFP to achieve efficient, cell-type specific labeling of the presynaptic compartment.

• Protocol was developed with the calyx of Held but is suitable for pre- and postsynaptic compartments of various neurons across multiple mammalian and invertebrate species.

Store-operated Ca²⁺ entry (SOCE) is a ubiquitous Ca²⁺ signaling modality mediated by Orai Ca²⁺ channels at the plasma membrane (PM) and the endoplasmic reticulum (ER) Ca²⁺ sensors STIM1/2. At steady state, Orai1 constitutively cycles between an intracellular compartment and the PM. Orai1 PM residency is modulated by its endocytosis and exocytosis rates. Therefore, Orai1 trafficking represents an important regulatory mechanism to define the levels of Ca²⁺ influx. Here, we present a protocol using the dually tagged YFP-HA-Orai1 with a cytosolic YFP and extracellular hemagglutinin (HA) tag to quantify Orai1 cycling rates. For measuring Orai1 endocytosis, cells expressing YFP-HA-Orai1 are incubated with mouse anti-HA antibody for various periods of time before being fixed and stained for surface Orai1 with Cy5-labeled anti-mouse IgG. The cells are fixed again, permeabilized, and stained with Cy3-labeled anti-mouse IgG to reveal anti-HA that has been internalized. To quantify Orai1 exocytosis rate, cells are incubated with anti-HA antibody for various incubation periods before being fixed, permeabilized, and then stained with Cy5-labeled anti-mouse IgG. The Cy5/YFP ratio is plotted over time and fitted with a mono-exponential growth curve to determine exocytosis rate. Although the described assays were developed to measure Orai1 trafficking, they are readily adaptable to other PM channels.

Key features

• Detailed protocols to quantify endocytosis and exocytosis rates of Orai1 at the plasma membrane that can be used in various cell lines.

• The endocytosis and exocytosis assays are readily adaptable to study the trafficking of other plasma membrane channels.

Graphical overview

In this article, we provide a method to isolate embryonic melanoblasts from reporter mouse strains. The mice from which these cells are isolated are bred into the ROSA26^mT/mG reporter background, which results in green fluorescent protein (GFP) expression in the targeted melanoblast population. These cells are isolated and purified by fluorescence-activated cell sorting using GFP fluorescence. We also provide a method to culture the purified melanoblasts for further analysis. This method yields > 99% purity melanoblasts specifically targeted, and can be used for a variety of studies, including gene expression, clonogenic experiments, and biological assays, such as viability, capacity for directional migration, or differentiation into melanin-producing melanocytic cells.

Graphical overview

Kidney diseases are a global health concern. Modeling of kidney disease for translational research is often challenging because of species specificities or the postmitotic status of kidney epithelial cells that make primary cultures, for example podocytes. Here, we report a protocol for preparing primary cultures of podocytes based on the isolation and in vitro propagation of immature kidney progenitor cells subsequently differentiated into mature podocytes. This protocol can be useful for studying physiology and pathophysiology of human kidney progenitors and to obtain differentiated podocytes for modeling podocytopathies and other kidney disorders involving podocytes.

Graphical overview

This protocol describes a method for detecting and quantifying calcium ions in the endoplasmic reticulum (ER) and cytoplasm of cultured cells using fluorescent reporter proteins and ImageJ software. Genetically engineered fluorescent reporter proteins, such as G-CEPIA1er and GCaMP6f, localize to intracellular regions of interest (i.e., ER and cytoplasm) and emit green fluorescence upon binding to calcium ions. In this way, the fluorescence brightness of cells transfected with expression vectors for these reporters reflects the calcium ion concentration in each intracellular region. Here, we describe procedures for observing cultured cells expressing these fluorescent reporters under a fluorescence microscope, analyzing the obtained image using the free image analysis software ImageJ (https://imagej.net/ij/index.html), and determining the average fluorescence brightness of multiple cells present in the image. The current method allows us to quickly and easily quantify calcium ions on an image containing multiple cells and to determine whether there are relative differences in intracellular calcium ion concentration among experiments with different conditions.

Key features

• Detection and quantification of calcium ions in the ER and cytoplasm using fluorescent reporter proteins

• Quick and easy verification of measurement results using ImageJ

• Simultaneous comparison between various experimental conditions (drug treatment, mutants, etc.)

For several decades, aging in Saccharomyces cerevisiae has been studied in hopes of understanding its causes and identifying conserved pathways that also drive aging in multicellular eukaryotes. While the short lifespan and unicellular nature of budding yeast has allowed its aging process to be observed by dissecting mother cells away from daughter cells under a microscope, this technique does not allow continuous, high-resolution, and high-throughput studies to be performed. Here, we present a protocol for constructing microfluidic devices for studying yeast aging that are free from these limitations. Our approach uses multilayer photolithography and soft lithography with polydimethylsiloxane (PDMS) to construct microfluidic devices with distinct single-cell trapping regions as well as channels for supplying media and removing recently born daughter cells. By doing so, aging yeast cells can be imaged at scale for the entirety of their lifespans, and the dynamics of molecular processes within single cells can be simultaneously tracked using fluorescence microscopy.

Key features

• This protocol requires access to a photolithography lab in a cleanroom facility.

• Photolithography process for patterning photoresist on silicon wafers with multiple different feature heights.

• Soft lithography process for making PDMS microfluidic devices from silicon wafer templates.

Due to technical limitations, research to date has mainly focused on the role of abiotic and biotic stress–signalling molecules in the aerial organs of plants, including the whole shoot, stem, and leaves. Novel experimental platforms including the dual-flow-RootChip (dfRC), PlantChip, and RootArray have since expanded this to plant-root cell analysis. Based on microfluidic platforms for flow stream shaping and force sensing on tip-growing organisms, the dfRC has further been expanded into a bi-directional dual‐flow‐RootChip (bi-dfRC), incorporating a second adjacent pair of inlets/outlet, enabling bi-directional asymmetric perfusion of treatments towards plant roots (shoot-to-root or root-to-shoot). This protocol outlines, in detail, the design and use of the bi-dfRC platform. Plant culture on chip is combined with guided root growth and controlled exposure of the primary root to solute changes. The impact of surface treatment on root growth and defence signals can be tracked in response to abiotic and biotic stress or the combinatory effect of both. In particular, this protocol highlights the ability of the platform to culture a variety of plants, such as Arabidopsis thaliana, Nicotiana benthamiana, and Solanum lycopersicum, on chip. It demonstrates that by simply altering the dimensions of the bi-dfRC, a broad application basis to study desired plant species with varying primary root sizes under microfluidics is achieved.

Key features

• Expansion of the method developed by Stanley et al. (2018a) to study the directionality of defence signals responding to localised treatments.

Description of a microfluidic platform allowing culture of plants with primary roots up to 40 mm length, 550 μm width, and 500 μm height.

Treatment with polyvinylpyrrolidone (PVP) to permanently retain the hydrophilicity of partially hydrophobic bi-dfRC microchannels, enabling use with surface-sensitive plant lines.

• Description of novel tubing array setup equipped with rotatable valves for switching treatment reagent and orientation, while live-imaging on the bi-dfRC.

Graphical overview

Current means to quantify cells, gene expression, and fibrosis of liver histological slides are not standardized in the research community and typically rely upon data acquired from a selection of random regions identified in each slide. As such, analyses are subject to selection bias as well as limited subsets of available data elements throughout the slide. A whole-slide analysis of cells and fibrosis would provide for a more accurate and complete quantitative analysis, along with minimization of intra- and inter-experimental variables. Herein, we present LiverQuant, a method for quantifying whole-slide scans of digitized histologic images to render a more comprehensive analysis of presented data elements. After loading images and preparing the project in the QuPath program, researchers are provided with one to two scripts per analysis that generate an average intensity threshold for their staining, automated tissue annotation, and downstream detection of their anticipated cellular matrices. When compared with two standard methodologies for histological quantification, LiverQuant had two significant advantages: increased speed and a 50-fold greater tissue area coverage. Using publicly available open-source code (GitHub), LiverQuant improves the reliability and reproducibility of experimental results while reducing the time scientists require to perform bulk analysis of liver histology. This analytical process is readily adaptable by most laboratories, requires minimal optimization, and its principles and code can be optimized for use in other organs.

Graphical overview

In vivo microscopy of plants with high-frequency imaging allows observation and characterization of the dynamic responses of plants to stimuli. It provides access to responses that could not be observed by imaging at a given time point. Such methods are particularly suitable for the observation of fast cellular events such as membrane potential changes. Classical measurement of membrane potential by probe impaling gives quantitative and precise measurements. However, it is invasive, requires specialized equipment, and only allows measurement of one cell at a time. To circumvent some of these limitations, we developed a method to relatively quantify membrane potential variations in Arabidopsis thaliana roots using the fluorescence of the voltage reporter DISBAC₂(3). In this protocol, we describe how to prepare experiments for agar media and microfluidics, and we detail the image analysis. We take an example of the rapid plasma membrane depolarization induced by the phytohormone auxin to illustrate the method. Relative membrane potential measurements using DISBAC₂(3) fluorescence increase the spatio-temporal resolution of the measurements and are non-invasive and suitable for live imaging of growing roots. Studying membrane potential with a more flexible method allows to efficiently combine mature electrophysiology literature and new molecular knowledge to achieve a better understanding of plant behaviors.

Key features

• Non-invasive method to relatively quantify membrane potential in plant roots.

• Method suitable for imaging seedlings root in agar or liquid medium.

• Straightforward quantification.