Cell Biology


Protocols in Current Issue
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0 Q&A 354 Views Apr 5, 2023

The developing cerebral cortex of mammals is generated from nascent pyramidal neurons, which radially migrate from their birthplace in the ventral part of the neural tube to the cortical surface. Subtle aberrations in this process may cause significant changes in cortical structure and lead to developmental neurological disorders. During pyramidal neuron migration, we recently showed that the migrating neuron, which bypasses its last preceding neuron, is critical for its proper positioning and contributes to cerebral cortex thickness. Studying this process requires an imaging system with single-cell resolution and a prolonged observation window. Therefore, we built a system to maintain an organotypic brain slice on the stage of a Leica SP5 confocal microscope, which facilitated high-resolution imaging over a 12-hour time-lapse observation period of cellular events during neuron migration. Here, we share our protocol along with guidelines for overcoming difficulties during the setup. This protocol facilitates the observation of, but is not limited to, neurodevelopmental and pathological processes occurring during neuron migration.

0 Q&A 924 Views Dec 20, 2022

The extracellular matrix (ECM) is a non-cellular network of macromolecules, which provides cells and tissues with structural support and biomechanical feedback to regulate cellular function, tissue tension, and homeostasis. Even subtle changes to ECM abundance, architecture, and organization can affect downstream biological pathways, thereby influencing normal cell and tissue function and also driving disease conditions. For example, in cancer, the ECM is well known to provide both biophysical and biochemical cues that influence cancer initiation, progression, and metastasis, highlighting the need to better understand cell–ECM interactions in cancer and other ECM-enriched diseases. Initial cell-derived matrix (CDM) models were used as an in vitro system to mimic and assess the physiologically relevant three-dimensional (3D) cell–ECM interactions. Here, we describe an expansion to these initial CDM models generated by fibroblasts to assess the effect of genetic or pharmacological intervention on fibroblast-mediated matrix production and organization. Additionally, we highlight current methodologies to quantify changes in the ultrastructure and isotropy of the resulting ECM and also provide protocols for assessing cancer cell interaction with CDMs. Understanding the nature and influence of these complex and heterogeneous processes can offer insights into the biomechanical and biochemical mechanisms, which drive cancer development and metastasis, and how we can target them to improve cancer outcomes.

0 Q&A 712 Views Nov 5, 2022

Mature B-cell lymphomas are highly dependent upon the protective lymphoid organ microenvironment for their growth and survival. Targeting integrin-mediated homing and retention of the malignant B cells in the lymphoid organs, using the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib, is a highly efficacious FDA-approved therapy for chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and Waldenström macroglobulinemia (WM). Unfortunately, a significant subset of patients is intrinsically resistant to ibrutinib or will develop resistance upon prolonged treatment. Here, we describe an unbiased functional genomic CRISPR-Cas9 screening method to identify novel proteins involved in B-cell receptor–controlled integrin-mediated adhesion, which provides novel therapeutic targets to overcome ibrutinib resistance. This screening method is highly flexible and can be easily adapted to identify cell adhesion–regulatory proteins and signaling pathways for other stimuli, adhesion molecules, and cell types.

Graphical abstract:

0 Q&A 767 Views Oct 5, 2022

Few models exist that allow for rapid and effective screening of anti-metastasis drugs. Here, we present a drug screening protocol utilizing gastrulation of zebrafish embryos for identification of anti-metastasis drugs. Based on the evidence that metastasis proceeds through utilizing the molecular mechanisms of gastrulation, we hypothesized that chemicals interrupting zebrafish gastrulation might suppress the metastasis of cancer cells. Thus, we developed a phenotype-based chemical screen that uses epiboly, the first morphogenetic movement in gastrulation, as a marker. The screen only needs zebrafish embryos and enables hundreds of chemicals to be tested in five hours by observing the epiboly progression of chemical-treated embryos. In the screen, embryos at the two-cell stage are firstly corrected and then developed to the sphere stage. The embryos are treated with a test chemical and incubated in the presence of the chemical until vehicle-treated embryos develop to the 90% epiboly stage. Finally, positive ‘hit’ chemicals that interrupt epiboly progression are selected by comparing epiboly progression of the chemical-treated and vehicle-treated embryos under a stereoscopic microscope. A previous study subjected 1,280 FDA-approved drugs to the screen and identified adrenosterone and pizotifen as epiboly-interrupting drugs. These were validated to suppress metastasis of breast cancer cells in mice models of metastasis. Furthermore, 11β-hydroxysteroid dehydrogenase 1 (HSD11β1) and serotonin receptor 2C (HTR2C), the primary targets of adrenosterone and pizotifen, respectively, promoted metastasis through induction of epithelial-mesenchymal transition (EMT). Therefore, this screen could be converted into a chemical genetic screening platform for identification of metastasis-promoting genes.

Graphical abstract:

0 Q&A 2006 Views Mar 5, 2022

Dozens of Mycoplasma species belonging to the class Mollicutes bind to solid surfaces through the organelle formed at a cell pole and glide in its direction by a unique mechanism. In Mycoplasma mobile, the fastest gliding species in Mycoplasma, the force for gliding is generated by ATP hydrolysis on an internal structure. However, the spatial and temporal behaviors of the internal structures in living cells were unclear. High-speed atomic force microscopy (HS-AFM) is a powerful method to monitor the dynamic behaviors of biomolecules and cells that can be captured while maintaining their active state in aqueous solution. In this protocol, we describe a method to detect their movements using HS-AFM. This protocol should be useful for the studies of many kinds of microorganisms.

Graphic abstract:

Scannnig Mycoplasma cell

0 Q&A 2852 Views Feb 20, 2022
The invasion of tumor cells into the neighboring blood vessels and lymph nodes is a vital step for distant metastasis. Traditionally, the invasive activity of growth factors (or the anti-invasive activity of drugs) is measured with the Boyden chamber assay. However, this assay has a few disadvantages like poor physiological relevance of transwell inserts and an inability to control chemokine gradients. The Boyden chamber assay is one of the most prevalent methods to measure the invasion of cancer cells. It would be advantageous to develop another assay that could validate the results of the Boyden chamber assay. With this in mind, our laboratory developed the spherical invasion assay (SIA) to measure the pro-invasive activity of human cancer cells. The SIA also circumvents some of the drawbacks of the Boyden chamber assay. The present manuscript measures the anti-invasive activity of the Src kinase inhibitor PP2 in A549 human non-small cell lung carcinoma (NSCLC) cells using the SIA. The SIA protocol is comprised of two steps. In the first step, A549 human NSCLC cells (treated or not with PP2) were mixed with Matrigel and seeded in the middle of an eight-well chamber slide. After 24 h, a second layer of Matrigel was overlaid over the first layer. Over the course of the next 24 h, the A549 cells invade from the primary to the secondary Matrigel layers. Subsequently, the cells are visualized by phase-contrast microscopy and the images obtained are quantified using ImageJ to calculate the anti-invasive activity of PP2 in A549 cells. The results of the SIA correlate well with Boyden chamber assays. The SIA may be adapted for multiple experimental designs, such as drug screening (to combat invasion and metastasis), measuring the pro-invasive activity of growth factors, and elucidating the signaling pathways underlying the pro-invasive/anti-invasive activity of biological modifiers.

Graphic abstract:

Diagrammatic illustration of the spherical invasion assay (Hurley et al., 2017).
A. The first layer is comprised of human cancer cells mixed in a 1:1 suspension with Phenol Red containing Matrigel (represented as LAYER 1 in the figure). After 24 h, the cancer cells grow and extend up to the boundary of this first layer. B. A second layer of 1:1 solution Phenol Red-free Matrigel, in Phenol Red-free RPMI (represented as LAYER 2 in the figure) is added on top of the first Matrigel spot. The cells are incubated for 24 h at 37°C. C. Over these 24 h, the cancer cells invade from the primary layer into the secondary Matrigel layer. The chamber slides are observed by phase-contrast microscopy. D. A representative photograph of the images obtained by the SIA is shown. The black arrow indicates the cancer cells invading into the second layer of Matrigel. The dotted line represents the interface between the two layers. The distance to which the cells have traveled (into the secondary Matrigel layer) is measured at ten sites (for each photograph) in a randomized double-blind fashion by three independent observers, using NIH ImageJ Version 1.47. This process is repeated for three separate photographic fields per sample.

0 Q&A 1561 Views Feb 5, 2022

Cell migration is a vital process in the development of multicellular organisms. When deregulated, it is involved in many diseases such as inflammation and cancer metastisation. Some cancer cells could be stimulated using chemoattractant molecules, such as growth factor Heregulin β1. They respond to the attractant or repellent gradients through a process known as chemotaxis. Indeed, chemotactic cell motility is crucial in tumour cell dissemination and invasion of distant organs. Due to the complexity of this phenomenon, the majority of available in vitro methods to study the chemotactic motility process have limitations and are mainly based on endpoint assays, such as the Boyden chamber assay. Nevertheless, in vitro time-lapse microscopy represents an interesting opportunity to study cell motility in a chemoattracting gradient, since it generates large volume image-based information, allowing the analysis of cancer cell behaviours. Here, we describe a detailed time-lapse imaging protocol, designed for tracking T47D human breast cancer cell line motility, toward a gradient of Heregulin β1 in a Dunn chemotaxis chamber assay. The protocol described here is readily adapted to study the motility of any adherent cell line, under various conditions of chemoattractant gradients and of pharmacological drug treatments. Moreover, this protocol could be suitable to study changes in cell morphology, and in cell polarity.

0 Q&A 1398 Views Feb 5, 2022

To identify causative substances for allergies to drugs or foods, the lymphocyte transformation test (LTT) is currently widely used as in vitro test, but its accuracy is not satisfactory. We have developed a novel method designated high-sensitivity allergy test (HiSAT) for determining allergy expression by measuring cell kinetics, using the chemotactic cells from non-allergic volunteers against a gradient field of cytokines released from immune cells when allergy develops. HiSAT requires a very small sample of 5 µL or less, and is applicable to three types of tests, depending on the situation in clinical practice: (i) diagnosis of the allergic expression, (ii) identification of the causative drug, and, in principle, (iii) pre-inspection.

Graphic abstract:

Schematic diagram of HiSAT. Serum from patients/subjects is used for rapid diagnosis in HiSAT. To identify the causative drug, the lymphocytes of interest are incubated with the candidate drug solution for 48 h to 72 h and then the culture supernatant is used in HiSAT. Before drug administration, it may possible to avoid the risk of allergies by performing pre-inspection, as well as the determination of the causative drug in HiSAT. A granulocyte-rich cell layer isolated from a non-allergic volunteer is used in HiSAT. Chemotactic cells migrate toward chemotactic factors in the test sample according to the concentration gradient. Cell kinetics (e.g., velocity or distance) are analyzed using sequential images of the test samples, and compared to the PHA-positive control.>

0 Q&A 2012 Views Jan 5, 2022

Blood endothelial cells (ECs) constitute the primary physical barrier to be crossed by circulating leukocytes, once attracted to a site of ongoing inflammation/infection. Upon a pro-inflammatory stimulus, such as tumor necrosis factor (TNF), ECs upregulate adhesion molecule expression to favor the adhesion and, subsequently, the transendothelial migration of the attracted lymphocytes. To address the ability of a cell to transmigrate through a monolayer of ECs, the classical transmigration assay is usually performed (Muller and Luscinskas, 2008). In the present protocol, adapted from Safuan et al. (2012), we describe an in vitro assay for assessing the functionality of the second step of the transendothelial migration, i.e., the firm adhesion of peripheral blood mononuclear cells (PBMCs) to ECs, under static conditions. By pre-incubating primary human umbilical cord ECs (HUVECs) with either innate lymphoid cell progenitors (ILCPs) or TNF, we were able to upregulate adhesion molecules on the EC surface. Then, by adding total PBMCs, we were able to both quantitatively and qualitatively analyze the cellular subtype and number of PBMCs that adhered to the pre-treated ECs. The important advantage of this technique is the possibility to perform functional studies on ECs biology since, differently from transwell-based strategies, it allows a good recovery of ECs at the end of the assay. Overall, this assay enables to interrogate how/if different stimulations/cell types can influence EC ability to retain PBMCs in vitro, under static conditions.

Graphic abstract:

The workflow of the Static Adhesion Assay.

0 Q&A 2638 Views May 5, 2021

Swarming – swift movement across a surface via flagella propulsion – is a unique property of many bacteria. The role of swarming, particularly among bacterial populations of the human gut microbiome, is not yet fully understood; although, it is becoming an area of increased scientific and clinical inquiry. To further characterize bacterial swarming in human health, an effective assay for swarming that utilizes complex material, such as fecal matter, is necessary. Until now, the vast majority of swarming assays have only been able to accommodate bacteria grown in culture, most often Pseudomonas. These assays tend to use a standard lysogenic broth (LB) agar medium; however, the reagents involved have not been tailored to the inoculation of complex material. In this paper, we offer a specialized protocol for eliciting the swarming of bacteria from frozen human fecal samples. We describe the simple, yet reproducible steps required to perform the assay, identifying an ideal volume of 7.5 μl for inoculation of material, as well as an ideal agar concentration of 0.4%. This protocol typically allows researchers to identify swarming within 24 h after incubation in a standard incubator.

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