Cell Biology


Protocols in Current Issue
Protocols in Past Issues
0 Q&A 283 Views May 5, 2024

The cation-independent mannose 6-phosphate receptors (CI-M6PR) bind newly synthesized mannose 6-phosphate (Man-6-P)-tagged enzymes in the Golgi and transport them to late endosomes/lysosomes, providing them with degradative functions. Following the cargo delivery, empty receptors are recycled via early/recycling endosomes back to the trans-Golgi network (TGN) retrogradely in a dynein-dependent motion. One of the most widely used methods for studying the retrograde trafficking of CI-M6PR involves employing the CD8α-CI-M6PR chimera. This chimera, comprising a CD8 ectodomain fused with the cytoplasmic tail of the CI-M6PR receptor, allows for labeling at the plasma membrane, followed by trafficking only in a retrograde direction. Previous studies utilizing the CD8α-CI-M6PR chimera have focused mainly on colocalization studies with various endocytic markers under steady-state conditions. This protocol extends the application of the CD8α-CI-M6PR chimera to live cell imaging, followed by a quantitative analysis of its motion towards the Golgi. Additionally, we present an approach to quantify parameters such as speed and track lengths associated with the motility of CD8α-CI-M6PR endosomes using the Fiji plugin TrackMate.

0 Q&A 623 Views Apr 20, 2024

In vivo brain imaging, using a combination of genetically encoded Ca2+ indicators and gradient refractive index (GRIN) lens, is a transformative technology that has become an increasingly potent research tool over the last decade. It allows direct visualisation of the dynamic cellular activity of deep brain neurons and glia in conscious animals and avoids the effect of anaesthesia on the network. This technique provides a step change in brain imaging where fibre photometry combines the whole ensemble of cellular activity, and multiphoton microscopy is limited to imaging superficial brain structures either under anaesthesia or in head-restrained conditions. We have refined the intravital imaging technique to image deep brain nuclei in the ventral medulla oblongata, one of the most difficult brain structures to image due to the movement of brainstem structures outside the cranial cavity during free behaviour (head and neck movement), whose targeting requires GRIN lens insertion through the cerebellum—a key structure for balance and movement. Our protocol refines the implantation method of GRIN lenses, giving the best possible approach to image deep extracranial brainstem structures in awake rodents with improved cell rejection/acceptance criteria during analysis. We have recently reported this method for imaging the activity of retrotrapezoid nucleus and raphe neurons to outline their chemosensitive characteristics. This revised method paves the way to image challenging brainstem structures to investigate their role in complex behaviours such as breathing, circulation, sleep, digestion, and swallowing, and could be extended to image and study the role of cerebellum in balance, movement, motor learning, and beyond.

Key features

• We developed a protocol that allows imaging from brainstem neurons and glia in freely behaving rodents.

• Our refined method of GRIN lenses implantation and cell sorting approach gives the highest number of cells with the least postoperative complications.

• The revised deep brainstem imaging method paves way to understand complex behaviours such as cardiorespiratory regulation, sleep, swallowing, and digestion.

• Our protocol can be implemented to image cerebellar structures to understand their role in key functions such as balance, movement, motor learning, and more.

Graphical overview

0 Q&A 486 Views Feb 5, 2024

Vertebrate embryogenesis is a highly dynamic process involving coordinated cell and tissue movements that generate the final embryonic body plan. Many of these movements are difficult to image at high resolution because they occur deep within the embryo along the midline, causing light scattering and requiring longer working distances. Here, we present an explant-based method to image transverse cross sections of living zebrafish embryos. This method allows for the capture of all cell movements at high-resolution throughout the embryonic trunk, including hard-to-image deep tissues. This technique offers an alternative to expensive or computationally difficult microscopy methods.

Key features

• Generates intact zebrafish explants with minimal tissue disturbance.

• Allows for live imaging of deep tissues normally obscured by common confocal microscopy techniques.

• Immobilizes tissues for extended periods required for time-lapse imaging.

• Utilizes readily available reagents and tools, which can minimize the time and cost of the procedure.

Graphical overview

0 Q&A 911 Views Jan 20, 2024

All living organisms require the division of a cell into daughter cells for their growth and maintenance. During cell division, both genetic and cytoplasmic contents are equally distributed between the two daughter cells. At the end of cell division, cytoplasmic contents and the plasma membrane are physically separated between the two daughter cells via a process known as cytokinesis. Hundreds of proteins and lipids involved in the cytokinetic process have been identified; however, much less is known about the mechanisms by which these molecules regulate cytokinesis, being therefore an intense area of current research. Male meiotic cytokinesis in Drosophila melanogaster testes has been shown to be an excellent model to study cytokinesis in vivo. Currently, several excellent protocols are available to study cytokinesis in Drosophila testes. However, improved methods are required to study cytokinesis under in vitro and ex vivo conditions. Here, we demonstrate a simple method to perform live imaging on individual spermatocyte cysts isolated from adult testes. We evaluate amenability of this in vitro method for treatment with pharmacological agents. We show that cytokinesis is strongly inhibited upon treatment with Dynasore, a dynamin inhibitor known to block clathrin-mediated endocytosis. In addition, we also demonstrate an ex vivo method to perform live imaging on whole mount adult testes on gas permeable membrane chambers. We believe the protocols described here are valuable tools to study cytokinetic mechanisms under various genetic and treatment conditions.

Key features

• In vitro method to study male meiotic cytokinesis in dissected spermatocyte cysts.

• In vitro method allows acute treatment with various pharmacological agents to study cytokinesis.

• Ex vivo method to image male meiosis cytokinesis in intact adult testes.

• Requires 15–60 min to set up and could be imaged up to 6–12 h.

Graphical overview

In vitro and ex vivo live imaging of male meiotic cytokinesis in adult Drosophila testes

0 Q&A 452 Views Dec 5, 2023

Neovascular diseases of the retina, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD), are proliferative retinopathies involving the growth of new blood vessels on the retina, which in turn causes impairment and potential loss of vision. A drawback of conventional angiogenesis assays is that they are not representative of the angiogenic processes in the retina. In the retina, the new blood vessels grow (from pre-existing blood vessels) and migrate into a non-perfused region of the eye including the inner limiting membrane of the retina and the vitreous, both of which contribute to vision loss. The Matrigel Duplex Assay (MDA) measures the migration of angiogenic capillaries from a primary Matrigel layer to a secondary Matrigel layer, which resembles the pathological angiogenesis in AMD and DR. The methodology of MDA is comprised of two steps. In the first step, the human retinal microvascular endothelial cells (HRMECs) are mixed with phenol red–containing Matrigel (in a 1:1 ratio) and seeded in the center of an 8-well chamber slide. After 24 h, a second layer of phenol red–free Matrigel is overlaid over the first layer. Over the course of the next 24 h, the HRMECs invade from the primary Matrigel layer to the secondary layer. Subsequently, the angiogenic sprouts are visualized by brightfield phase contrast microscopy and quantified by ImageJ software. The present manuscript measures the angiogenesis-inhibitory activity of the Src kinase inhibitor PP2 in primary HRMECs using the MDA. The MDA may be used for multiple applications like screening anti-angiogenic drugs, measuring the pro-angiogenic activity of growth factors, and elucidating signaling pathways underlying retinal angiogenesis in normal and disease states.

Graphical overview

0 Q&A 289 Views Nov 5, 2023

Pancreatic islet β cells preferentially secrete insulin toward the plasma membrane, making contact with the capillary extracellular matrix (ECM). Isolated islets separated from the exocrine acinar cells are the best system for cell biology studies of primary β cells, whereas isolated islets lose their capillary network during ex vivo culture. Providing the appropriate extracellular signaling by attaching islets to vascular ECM-coated surfaces can restore the polarized insulin secretion toward the ECM. The guided secretion toward ECM-coated glass coverslips provides a good model for recording insulin secretion in real time to study its regulation. Additionally, β cells attached to the ECM-coated coverslips are suitable for confocal live imaging of subcellular components including adhesion molecules, cytoskeleton, and ion channels. This procedure is also compatible for total internal reflection fluorescence (TIRF) microscopy, which provides optimal signal-to-noise ratio and high spatial precision of structures close to the plasma membrane. In this article, we describe the optimized protocol for vascular ECM-coating of glass coverslips and the process of attachment of isolated mouse islets on the coverslip. This preparation is compatible with any high-resolution microscopy of live primary β cells.

Key features

• Optimized coating procedure to attach isolated islets, compatible for both confocal and TIRF microscopy.

• The ECM-coated glass coverslip functions as the artificial capillary surface to guide secretion toward the coated surface for optimal imaging of secretion events.

• Shows the process of islets attachment to the ECM-coated surface in a 6-day ex vivo culture.

Graphical overview

0 Q&A 357 Views Aug 5, 2023

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.

0 Q&A 682 Views Jul 20, 2023

Intestinal intraepithelial lymphocytes (IEL) are a numerous population of T cells located within the epithelium of the small and large intestines, being more numerous in the small intestine (SI). They surveil this tissue by interacting with epithelial cells. Intravital microscopy is an important tool for visualizing the patrolling activity of IEL in the SI of live mice. Most IEL express CD8α; therefore, here we describe an established protocol of intravital imaging that tracks lymphocytes labeled with a CD8α-specific monoclonal antibody in the SI epithelium of live mice. We also describe data acquisition and quantification of the movement metrics, including mean speed, track length, displacement length, and paths for each CD8α+ IEL using the available software. The intravital imaging technique for measuring IEL movement will provide a better understanding of the role of IEL in homeostasis and protection from injury or infection in vivo.

0 Q&A 1075 Views Jul 20, 2023

Embryonic development is a complex process integrating cell fate decisions and morphogenesis in a spatiotemporally controlled manner. Previous studies with model organisms laid the foundation of our knowledge on post-implantation development; however, studying mammalian embryos at this stage is a difficult and laborious process. Early attempts to recapitulate mammalian development in vitro begun with embryoid bodies (EBs), in which aggregates of mouse embryonic stem cells (mESCs) were shown to differentiate into spatially arranged germ layers. A more revised version of EBs, gastruloids, improved the germ layer differentiation efficiency and demonstrated cell fate patterning on multiple axes. However, gastruloids lack anterior neural progenitors that give rise to brain tissues in the embryo. Here, we report a novel culture protocol to coax mESCs into post-implantation epiblast-like (EPI) aggregates in high throughput on bioengineered microwell arrays. We show that upon inhibition of the Wnt signaling pathway, EPI aggregates establish an extended axial patterning, leading to co-derivation of anterior neural progenitors and posterior tissues. Our approach is amenable to large-scale studies aimed at identifying novel regulators of gastrulation and anterior neural development that is currently out of reach with existing embryoid models. This work should contribute to the advancement of the nascent field of synthetic embryology, opening up exciting perspectives for various applications of pluripotent stem cells in disease modeling and tissue engineering.

Key features

• A new gastruloid culture system to model post-implantation mouse embryonic development in vitro

• High-throughput formation of epiblast-like aggregates on hydrogel microwells

• Builds upon conventional gastruloid cultures and provides insight into the role of Wnt signaling for the formation of anterior neural tissues

Graphical overview

0 Q&A 1030 Views Jul 20, 2023

Loss of plasma membrane lipid asymmetry contributes to many cellular functions and responses, including apoptosis, blood coagulation, and cell fusion. In this protocol, we describe the use of fluorescently labeled annexin V to detect loss of lipid asymmetry in the plasma membrane of adherent living cells by fluorescence microscopy. The approach provides a simple, sensitive, and reproducible method to detect changes in lipid asymmetry but is limited by low sample throughput. The protocol can also be adapted to other fluorescently labeled lipid-binding proteins or peptide probes. To validate the lipid binding properties of such probes, we additionally describe here the preparation and use of giant unilamellar vesicles as simple model membrane systems that have a size comparable to cells.

Key features

• Monitoring loss of lipid asymmetry in the plasma membrane via confocal microscopy.

• Protocol can be applied to any type of cell that is adherent in culture, including primary cells.

• Assay can be adapted to other fluorescently labeled lipid-binding proteins or peptide probes.

• Giant unilamellar vesicles serve as a tool to validate the lipid binding properties of such probes.

Graphical overview

Imaging the binding of fluorescent annexin V to adherent mammalian cells and giant vesicles by confocal microscopy. Annexin V labeling is a useful method for detecting a loss of plasma membrane lipid asymmetry in cells (top image, red); DAPI can be used to identify nuclei (top image, blue). Giant vesicles are used as a tool to validate the lipid binding properties of annexin V to anionic lipids (lower image, red).

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