Cell Culture Infectivity to Assess Chlorine Disinfection of Cryptosporidium Oocysts in Water.
Abstract This chapter provides a detailed protocol to assess disinfection efficacy of chlorine against Cryptosporidium oocysts including the core chlorine disinfection assay, the in vitro cell culture infectivity assay, and microscopy analysis and data interpretation. PMID: 31452168 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - August 29, 2019 Category: Molecular Biology Authors: Murphy JL, Arrowood MJ Tags: Methods Mol Biol Source Type: research

High-Content Screening for Cryptosporidium Drug Discovery.
Abstract High-content screening (HCS) is a cell-based type of phenotypic screening that combines multiple simultaneous readouts with a high level of throughput. A particular benefit of this form of screening for drug discovery is the ability to perform the interrogation in a biologically relevant system. This approach has greatly advanced the field of drug discovery for cryptosporidiosis, a diarrheal disease caused by protozoan parasites of Cryptosporidium spp. These parasites are obligate intracellular parasites and cannot be cultured in vitro without the support of a host cell, limiting the options for potential...
Source: Mol Biol Cell - August 29, 2019 Category: Molecular Biology Authors: Love MS, McNamara CW Tags: Methods Mol Biol Source Type: research

High-Throughput Screening of Drugs Against the Growth of Cryptosporidium parvum In Vitro by qRT-PCR.
Abstract An effective method to quantify the parasite loads is the key to the evaluation of anti-cryptosporidial drug efficacy in vitro. However, high-throughput screening (HTS) of drugs against Cryptosporidium parvum in vitro was impractical by the labor-intensive traditional assays. Here we describe a simplified quantitative RT-PCR assay suitable for HTS of compounds and for evaluating drug efficacy against the growth of C. parvum in vitro. PMID: 31452170 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - August 29, 2019 Category: Molecular Biology Authors: Zhang H, Zhu G Tags: Methods Mol Biol Source Type: research

In Vitro Culture of Cryptosporidium parvum Using Stem Cell-Derived Intestinal Epithelial Monolayers.
We describe here a method to create primary intestinal epithelial cell monolayers that support long-term C. parvum growth. Monolayers were derived from mouse intestinal stem cells grown as spheroids and plated onto transwells, allowing for separate apical and basolateral compartments. In the apical chamber, the cell growth medium was removed to create an "air-liquid interface" that enhanced host cell differentiation and supported long-term C. parvum growth. The use of primary intestinal cells to grow C. parvum in vitro will be a valuable tool for studying host-parasite interactions using a convenient in vitro mod...
Source: Mol Biol Cell - August 29, 2019 Category: Molecular Biology Authors: Wilke G, Wang Y, Ravindran S, Stappenbeck T, Witola WH, Sibley LD Tags: Methods Mol Biol Source Type: research

Two- and Three-Dimensional Bioengineered Human Intestinal Tissue Models for Cryptosporidium.
Abstract Conventional cell cultures utilizing transformed or immortalized cell lines or primary human epithelial cells have played a fundamental role in furthering our understanding of Cryptosporidium infection. However, they remain inadequate with respect to their inability to emulate in vivo conditions, support long-term growth, and complete the life cycle of the parasite. Previously, we developed a 3D silk scaffold-based model using transformed human intestinal epithelial cells (IECs). This model supported C. parvum infection for up to 2 weeks and resulted in completion of the life cycle of the parasite. H...
Source: Mol Biol Cell - August 29, 2019 Category: Molecular Biology Authors: Cardenas D, Bhalchandra S, Lamisere H, Chen Y, Zeng XL, Ramani S, Karandikar UC, Kaplan DL, Estes MK, Ward HD Tags: Methods Mol Biol Source Type: research

The Golgi Apparatus in Polarized Neuroepithelial Stem Cells and Their Progeny: Canonical and Noncanonical Features.
Abstract Neurons forming the central nervous system are generated by neural stem and progenitor cells, via a process called neurogenesis (Götz and Huttner, Nat Rev Mol Cell Biol, 6:777-788, 2005). In this book chapter, we focus on neurogenesis in the dorsolateral telencephalon, the rostral-most region of the neural tube, which contains the part of the central nervous system that is most expanded in mammals (Borrell and Reillo, Dev Neurobiol, 72:955-971, 2012; Wilsch-Bräuninger et al., Curr Opin Neurobiol 39:122-132, 2016). We will discuss recent advances in the dissection of the cell biological mechanism...
Source: Mol Biol Cell - August 24, 2019 Category: Molecular Biology Authors: Taverna E, Huttner WB Tags: Results Probl Cell Differ Source Type: research

An Overview of Drug Delivery Systems.
Abstract This is an overview of the current drug delivery systems (DDSs) starting with various routes of drug administration. Various drug formulations are then described as well as devices used for drug delivery and targeted drug delivery. There has been a considerable increase in the number of new biotechnology-based therapeutics. Most of these are proteins and peptides, and their delivery present special challenges. Cell and gene therapies are sophisticated methods of delivery of therapeutics. Nanoparticles are important for refining drug delivery. In addition to being vehicles for drug delivery, nanoparticles ...
Source: Mol Biol Cell - August 24, 2019 Category: Molecular Biology Authors: Jain KK Tags: Methods Mol Biol Source Type: research

Role of Nanobiotechnology in Drug Delivery.
Abstract This chapter is a brief overview of use of nanobiotechnology in drug delivery. Several types of nanoparticles are available. Nanoparticulate formulations of normally used drugs have increased efficacy due to improved absorption and require lower dosage with less side effects than standard formulations. Nanobiotechnology also facilitates targeted drug delivery of anticancer drugs, which is important for the management of cancer. Nanoparticles also facilitate crossing of biological barriers in the human body for drug delivery to targeted organs, for example, crossing the blood-brain barrier to reach the bra...
Source: Mol Biol Cell - August 24, 2019 Category: Molecular Biology Authors: Jain KK Tags: Methods Mol Biol Source Type: research

A Method for Targeted Nonviral siRNA Delivery in Cancer and Inflammatory Diseases.
Abstract Small interfering RNA (siRNA)-based therapy has been subject of intense research since the discovery of RNA interference (RNAi), providing a tool to potentially silence any chosen gene. Nevertheless, efficient delivery still presents a major hurdle to translating this promising technology into medical practice. Here, we describe a straightforward method to prepare and characterize an effective delivery system consisting of low-molecular-weight polyethylenimine (PEI) and transferrin (Tf). Tf-PEI polyplexes are not only able to successfully transport and protect the sensitive nucleic acid payload from degra...
Source: Mol Biol Cell - August 24, 2019 Category: Molecular Biology Authors: Kandil R, Xie Y, Mehta A, Merkel O Tags: Methods Mol Biol Source Type: research

Microfluidic Manufacturing of Multitargeted PLGA/PEG Nanoparticles for Delivery of Taxane Chemotherapeutics.
Abstract Taxane chemotherapeutics have played a key role in the treatment of various types of cancer throughout the past years. However, the drawbacks inherent to the pharmaceutical formulation of taxanes are still a reality and mainly due to the low aqueous solubility of these medicines, as well as to the nontargeted therapy and consequent side effects. Nanoparticles (NPs) of poly(lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG) have sparked broad interest in this field and demonstrated capacity of improving taxanes' formulation. If, in one hand, the PLGA core of these NPs is able to solubilize drugs...
Source: Mol Biol Cell - August 24, 2019 Category: Molecular Biology Authors: Martins C, Sarmento B Tags: Methods Mol Biol Source Type: research

Targeted siRNA Delivery Using Lipid Nanoparticles.
Abstract Efficient intracellular delivery of small-interfering ribonucleic acid (siRNA) to the target organ or tissues in the body is assumed as the main hurdle for a widespread use of siRNAs in the clinics. Solid lipid-based nanoparticles (SLNs) and derivatives can potentially fit this purpose by enabling to overcome the extracellular and intracellular physiological barriers affecting the delivery. For that, rational formulations and rational process designs are needed. This chapter addresses a comprehensive description and critical appraisal of the main production methods of this particular type of lipid nanopar...
Source: Mol Biol Cell - August 24, 2019 Category: Molecular Biology Authors: Jorge A, Pais A, Vitorino C Tags: Methods Mol Biol Source Type: research

Construction of a Macrophage-Targeting Bio-nanocapsule-Based Nanocarrier.
Abstract The construction protocol of bio-nanocapsule (BNC)-based nanocarriers, named GL-BNC and GL-virosome, for targeted drug delivery to macrophages is described here. First, genes encoding the Streptococcus sp. protein G-derived C2 domain (binds to IgG Fc) and Finegoldia magna protein L-derived B1 domain (binds to Igκ light chain) are prepared by PCR amplification. Subsequently, the genes encoding hepatic cell-specific binding domain of hepatitis B virus envelope L protein are replaced by these PCR products. The expression plasmid for this fused gene (encoding GL-fused L protein) can be used to transform...
Source: Mol Biol Cell - August 24, 2019 Category: Molecular Biology Authors: Li H, Somiya M, Tatematsu K, Kuroda S Tags: Methods Mol Biol Source Type: research

Production and Characterization of Chemically Cross-Linked Collagen Scaffolds.
is DI Abstract Chemical cross-linking of collagen-based devices is used as a means of increasing the mechanical stability and control the degradation rate upon implantation. Herein, we describe techniques to produce cross-linked with glutaraldehyde (GTA; amine terminal cross-linker), 4-arm polyethylene glycol succinimidyl glutarate (4SP; amine terminal cross-linker), diphenyl phosphoryl azide (DPPA; carboxyl terminal cross-linker), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; carboxyl terminal cross-linker) collagen films. In addition, we provide protocols to characterize the biophysical (swelling), bio...
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Sallent I, Capella-Monsonís H, Zeugolis DI Tags: Methods Mol Biol Source Type: research

Monitoring for Epigenetic Modifications at the FMR1 Locus.
Abstract The vast majority of fragile X affected patients do not transcribe FMR1 due to a CGG repeat expansion in the 5'-untranslated region of the FMR1 gene. When the CGGs considerably expand, it elicits abnormal DNA methylation and histone modifications, which are responsible for FMR1 transcriptional silencing. In this chapter, we describe in detail two commonly used protocols for monitoring the epigenetic state of the FMR1 gene that bypass the difficulty in directly analyzing the CGGs. One protocol is for accurately measuring DNA methylation levels and the other is for profiling histone modifications. PMID...
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Epsztejn-Litman S, Eiges R Tags: Methods Mol Biol Source Type: research

Assays for Determining Repeat Number, Methylation Status, and AGG Interruptions in the Fragile X-Related Disorders.
We describe here assays that we use in our laboratory to assess these parameters. Our assays are PCR-based and include one for repeat size that can also be used to assess the extent of methylation and a related assay that allows the AGG interruption pattern to be reliably determined even in women. A second more quantitative methylation assay is also described. We also describe our method for cloning of repeats to generate the reference standards necessary for the accurate determination of repeat number and AGG interruption status. PMID: 30900174 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Hayward BE, Usdin K Tags: Methods Mol Biol Source Type: research

One-Step Generation of Seamless Luciferase Gene Knockin Using CRISPR/Cas9 Genome Editing in Human Pluripotent Stem Cells.
Abstract Human pluripotent stem cells (hPSCs) offer powerful platforms for studying mechanisms of human diseases and for evaluating potential treatments. Genome editing, particularly the CRISPR/Cas9-based method, is highly effective for generating cell and animal models to study genetic human diseases. However, the procedure for generating gene-edited hPSCs is laborious, time consuming and unintentional genetic changes may confound the consequent experiments and conclusions. Here we describe one-step knockin of the NanoLuc luciferase gene (Nluc) to the fragile X syndrome gene, FMR1, in a human embryonic stem cell ...
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Li M, Hunt JFVS, Bhattacharyya A, Zhao X Tags: Methods Mol Biol Source Type: research

Modeling FXS with Mouse Neural Progenitors.
;n ML Abstract The neurosphere assay is a widely used method to culture neural precursor cells (NPCs), which include mixed populations of neural stem and progenitor cells, from the mammalian central nervous system. Fmr1-knockout (KO) mice generated to model fragile X syndrome (FXS) recapitulate the major phenotype of FXS. Neurosphere differentiation of cortical progenitors derived from brains of Fmr1-KO mice has been shown to reflect disordered mechanisms during cortical development in FXS in vivo. The cellular composition of neurospheres is heterogeneous, but robust FXS-specific alterations can be identified when...
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Peteri UK, Castrén ML Tags: Methods Mol Biol Source Type: research

Using Human Neural Progenitor Cell Models to Conduct Large-Scale Drug Screens for Neurological and Psychiatric Diseases.
Abstract High-throughput drug screen (HTS) has become a viable approach for new treatment discovery in human diseases. Advances in gene editing technology and human pluripotent stem cell differentiation techniques have expanded the capability of HTS to identify potential treatments for human diseases of the central nervous system. Here, we describe techniques to use a human patient-derived neural progenitor cell luciferase reporter line to screen a large small molecule library. PMID: 30900177 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Hunt JFVS, Li M, Zhao X, Bhattacharyya A Tags: Methods Mol Biol Source Type: research

Modeling FXS: Human Pluripotent Stem Cells and In Vitro Neural Differentiation.
Abstract In fragile X syndrome (FXS) embryos FMRP is widely expressed during early stages of embryogenesis however it is inactivated by the end of the first trimester. In the same manner, human embryonic stem cell (hESC) lines from FXS blastocysts, bearing the full CGG expansion mutation, express FMRP in their pluripotent stage and in neurons derived following in vitro differentiation, FMR1 is completely silenced. Therefore, in vitro neural differentiation of FX-hESC lines serves as a uniquely valuable model system to study the developmental mechanisms underlying FXS, together with the proper differentiation proto...
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Kuznitsov-Yanovsky L, Mayshar Y, Ben-Yosef D Tags: Methods Mol Biol Source Type: research

Induced Neurons for the Study of Neurodegenerative and Neurodevelopmental Disorders.
Abstract Patient-derived or genomically modified human induced pluripotent stem cells (iPSCs) offer the opportunity to study neurodevelopmental and neurodegenerative disorders. Overexpression of certain neurogenic transcription factors (TFs) in iPSCs can induce efficient differentiation into homogeneous populations of the disease-relevant neuronal cell types. Here we provide protocols for genomic manipulations of iPSCs by CRISPR/Cas9. We also introduce two methods, based on lentiviral delivery and the piggyBac transposon system, to stably integrate neurogenic TFs into human iPSCs. Furthermore, we describe the TF-m...
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Sauter EJ, Kutsche LK, Klapper SD, Busskamp V Tags: Methods Mol Biol Source Type: research

Imaging of Somatic Ca2+ Transients in Differentiated Human Neurons.
Abstract Calcium is a major regulator of neuronal activity and calcium signaling is critically important for normal neuronal function. Ca imaging is a well-established tool for studying neuronal function and ongoing spontaneous Ca2+ transients are a good indicator of neuronal maturity. There are various indicators available today, differing by their sensitivity, spectra, and loading method. Here we present a method for measurement of Ca2+ transients in neurons using two different Ca2+ indicators, Oregon Green BAPTA-1 and GCaMP6. PMID: 30900180 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Vertkin I, Ben-Yosef D Tags: Methods Mol Biol Source Type: research

Patch-Clamp Recordings from Human Embryonic Stem Cells-Derived Fragile X Neurons.
Abstract Performing electrophysiological recordings from human neurons that have been differentiated in vitro, as compared to primary cultures, raises many challenges. However, patch-clamp recording from neurons derived from stem cells provides an abundance of valuable information, reliably and fast. Here, we describe a protocol that is used successfully in our lab for recording from both control and Fragile X neurons, derived in vitro from human embryonic stem cells. PMID: 30900181 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Telias M, Segal M Tags: Methods Mol Biol Source Type: research

Fragile X Syndrome Pre-Clinical Research: Comparing Mouse- and Human-Based Models.
Abstract Despite almost 30 years of biomedical research, a treatment or cure for fragile X syndrome (FXS) is not yet available. The reasons behind this are varied, and among them are discrepancies in both research methodologies and research models. For many years, the fmr1 knockout mouse model dominated the field, and was used to draw important conclusions. The establishment of FXS-human cellular models called these conclusions into question, showing conflicting evidence. Discrepancies in FXS research, between mouse and human, might arise from differences inherent to each species, and from the use of dif...
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Telias M Tags: Methods Mol Biol Source Type: research

Costunolide reduces glycolysis-associated activation of hepatic stellate cells via inhibition of hexokinase-2.
Conclusions: Our results show that costunolide can suppress HSC activation, and this is associated with inhibition of HK2, which blocks aerobic glycolysis. This suggests that costunolide is an antifibrotic candidate with potential for further development. PMID: 31428167 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Ban D, Hua S, Zhang W, Shen C, Miao X, Liu W Tags: Cell Mol Biol Lett Source Type: research

Current prevalence status of gastric cancer and recent studies on the roles of circular RNAs and methods used to investigate circular RNAs.
This study reviews current prevalence of gastric cancer in the word and China, as well as the characteristics and functions of circRNA and common laboratory detection methods involving circRNA in gastric cancer. PMID: 31428168 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Jiang F, Shen X Tags: Cell Mol Biol Lett Source Type: research

Functional Analyses of Embryonic Cerebrospinal Fluid Proteins.
Abstract The embryonic cerebrospinal fluid (eCSF) influences neuroepithelial cell behavior, affecting proliferation, differentiation, and survival. One major question to resolve in the field is to precisely describe the eCSF molecules responsible and to understand how these molecules interact in order to exert their functions. Here we describe an in vitro protocol to analyze the influence of eCSF components on neuroepithelium development. PMID: 31432405 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Caprile T, Lamus F, Alonso MI, Montecinos H, Gato A Tags: Methods Mol Biol Source Type: research

SWATH Mass Spectrometry Applied to Cerebrospinal Fluid Differential Proteomics: Establishment of a Sample-Specific Method.
Abstract Mass spectrometry (MS) has become the gold standard method for proteomics by allowing the simultaneous identification and/or quantification of thousands of proteins of a given sample. Over time, mass spectrometry has evolved into newer quantitative approaches with increased sensitivity and accuracy, such as the sequential windows acquisition of all theoretical fragment-ion spectra (SWATH)-MS approach. Moreover, in the past few years, some improvements were made in the SWATH-acquisition algorithm, allowing the design of sample-customized acquisition methods by adjusting the Q1 windows' width...
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Anjo SI, Santa C, Manadas B Tags: Methods Mol Biol Source Type: research

Untargeted Metabolomics Relative Quantification by SWATH Mass Spectrometry Applied to Cerebrospinal Fluid.
Abstract Cerebrospinal fluid (CSF) has been considered the key source for the search of biomarkers, in particular for neurological diseases, such as Alzheimer's and Parkinson's disease, since it reflects the state of the central nervous system (CNS). Finding biomarkers in the earliest stages of neurodegenerative diseases has become imperative, since, at the moment, there are no drugs that can reverse these pathological processes. Untargeted metabolomics analysis by liquid chromatography combined with SWATH-MS relative quantification is an emerging approach to search for potential biomarkers. In this chapter, we de...
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Mendes VM, Coelho M, Manadas B Tags: Methods Mol Biol Source Type: research

Characterization of Alzheimer's Disease Micro-RNA Profile in Exosome-Enriched CSF Samples.
uan P Abstract Micro-RNAs (miRNAs) are small endogenous noncoding ribonucleic acids that modulate gene expression at a post-transcriptional level. miRNAs have been postulated as potential biomarkers and therapeutic targets in a wide list of human diseases including cancer, autoimmune, cardiovascular, and neurodegenerative diseases. miRNAs are secreted by the cells into exosomes. These are small cell-derived membrane vesicles that can be isolated from many body fluids including urine, saliva, blood, and cerebrospinal fluid (CSF). Exosomes contain a variety of proteins and noncoding RNAs and seem to play an importan...
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Riancho J, Santurtun A, Sánchez-Juan P Tags: Methods Mol Biol Source Type: research

Automated Quantitative Analysis of Mitochondrial Morphology.
Abstract Mitochondria are dynamic organelles that in most cells behave as a dynamic network and can change their biogenesis and structure depending on the cell needs or as a response to different conditions. Analyzing the architecture of mitochondria is determinant to describe their state and function. In this chapter, image processing techniques are applied in a workflow manner to segment the mitochondrial network and extract the most relevant parameters that enable an accurate morphology analysis. This workflow is programmed with ImageJ macro language and can be applied to automatically analyze multiple cells fr...
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Bosch A, Calvo M Tags: Methods Mol Biol Source Type: research

Triple-Colocalization Approach to Assess Traffic Patterns and Their Modulation.
Abstract Confocal microscopy permits the analysis of the subcellular distribution of proteins. Colocalization between target proteins and specific markers of differential cell compartments provides an efficient approach to studying protein traffic. In this chapter, we describe an automated method to denoise confocal microscopy images and assess the colocalization of their stainings using ImageJ software. As a step further from conventional single colocalization measurements, in the proposed method, we analyze stacks of three different stainings using two-by-two comparisons. To demonstrate the reliability and usefu...
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Sastre D, Estadella I, Bosch M, Felipe A Tags: Methods Mol Biol Source Type: research

Cell Proliferation High-Content Screening on Adherent Cell Cultures.
sa JF Abstract Pulse-chase experiments using 5-bromo-2'-deoxyuridine (BrdU), or the more recent EdU (5-etynil-2'-deoxyuridine), enable the identification of cells going through S phase. This chapter describes a high-content proliferation assay pipeline for adherent cell cultures. High-throughput imaging is followed by high-content data analysis using a non-supervised ImageJ macroinstruction that segments the individual nuclei, determines the nucleoside analogue absence/presence, and measures the signal of up to two additional nuclear markers. Based upon the specific combination with proliferation-specific protein ...
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Carrillo-Barberà P, Morante-Redolat JM, Pertusa JF Tags: Methods Mol Biol Source Type: research

HCS Methodology for Helping in Lab Scale Image-Based Assays.
Abstract High-content screening (HCS) automates image acquisition and analysis in microscopy. This technology considers the multiple parameters contained in the images and produces statistically significant results. The recent improvements in image acquisition throughput, image analysis, and machine learning (ML) have popularized this kind of experiments, emphasizing the need for new tools and know-how to help in its design, analysis, and data interpretation. This chapter summarizes HCS recommendations for lab scale assays and provides both macros for HCS-oriented image analysis and user-friendly tools for data mi...
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Soriano J, Mata G, Megias D Tags: Methods Mol Biol Source Type: research

Filopodia Quantification Using FiloQuant.
Abstract Filopodia are fingerlike membrane protrusions that are extended by cells in vitro and in vivo. Due to important roles in sensing the extracellular microenvironment, filopodia and filopodia-like protrusions have been implicated in numerous biological processes including epithelial sheet zippering in development and wound healing and in cancer progression. Recently, there has been an explosion in the number of software available to analyze specific features of cell protrusions with the aim of gaining mechanistic insights into the action of filopodia and filopodia-like structures. In this methods chapter, we...
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Jacquemet G, Hamidi H, Ivaska J Tags: Methods Mol Biol Source Type: research

Coincidence Analysis of Molecular Dynamics by Raster Image Correlation Spectroscopy.
Abstract The dynamics of cellular processes is a crucial aspect to consider when trying to understand cell function, particularly with regard to the coordination of complex mechanisms involving extensive molecular networks in different cell compartments. Thus, there is an urgent demand of methodologies able to obtain accurate spatiotemporal information on molecular dynamics in live cells. Different variants based on fluorescence correlation spectroscopy have been used successfully in the analysis of protein diffusion and complex or aggregation status. However, the available approaches are limited when simultaneous...
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Moreno DF, Aldea M Tags: Methods Mol Biol Source Type: research

3D Tracking of Migrating Cells from Live Microscopy Time-Lapses.
Abstract With rapidly advancing microscopy techniques for live cell imaging, we are now able to image groups of migrating cells in many different in vivo contexts. However, as the resulting data sets become larger and more complex, following the behavior of these cells and extracting accurate quantitative data become increasingly challenging. Here we present a protocol for carrying out accurate automated tracking of cells moving over time in 3D, implemented as custom-built macro scripts for ImageJ. As opposed to many generic tracking workflows, the workflow we propose here accounts for the overall movement of the ...
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Tosi S, Campbell K Tags: Methods Mol Biol Source Type: research

A Cell Segmentation/Tracking Tool Based on Machine Learning.
Abstract The ability to gain quantifiable, single-cell data from time-lapse microscopy images is dependent upon cell segmentation and tracking. Here, we present a detailed protocol for obtaining quality time-lapse movies and introduce a method to identify (segment) and track cells based on machine learning techniques (Fiji's Trainable Weka Segmentation) and custom, open-source Python scripts. To provide a hands-on experience, we provide datasets obtained using the aforementioned protocol. PMID: 31432490 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Deter HS, Dies M, Cameron CC, Butzin NC, Buceta J Tags: Methods Mol Biol Source Type: research

2D + Time Object Tracking Using Fiji and ilastik.
We describe these two methods in a step-by-step fashion, using as example a time-lapse microscopy movie of HeLa cells. PMID: 31432491 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - August 22, 2019 Category: Molecular Biology Authors: Urru A, González Ballester MA, Zhang C Tags: Methods Mol Biol Source Type: research

An Introduction to LFA-1/ICAM-1 Interactions in T-Cell Motility.
Abstract The inherent ability of T-cells to migrate is critical for a fully functional immune system, both in normal immune surveillance and for mounting an adaptive immune response. At the same time, inappropriate trafficking of T-cells can be a pathological factor for immune-mediated or chronic inflammatory diseases. T-cell motility is critically dependent on a series of ligand-receptor interactions, a precisely regulated intracellular signaling, an involvement of adaptor proteins, and dynamic remodeling of the cytoskeletal systems. The leukocyte integrin LFA-1 receptor present on T-cells binds to the ligand int...
Source: Mol Biol Cell - August 21, 2019 Category: Molecular Biology Authors: Verma NK, Kelleher D Tags: Methods Mol Biol Source Type: research

Isolation of Human Peripheral Blood T-Lymphocytes.
Abstract Peripheral blood is the most common source of T-lymphocytes for in vitro culture. Here, we present a simple and standardized method for small- or large-scale isolation of viable T-lymphocytes and other mononuclear cells from fresh peripheral blood or buffy coat blood samples using the density gradient centrifugation. T-cells obtained using the protocol described here can be used for a variety of downstream analysis, including cellular, molecular, and functional assays. PMID: 30610593 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - August 21, 2019 Category: Molecular Biology Authors: Kizhakeyil A, Ong ST, Fazil MHUT, Chalasani MLS, Prasannan P, Verma NK Tags: Methods Mol Biol Source Type: research

A Laboratory Model to Study T-Cell Motility.
Abstract Regulated migration of T-lymphocytes through high endothelial venules and secondary lymphoid organs is necessary for an adaptive immune response. Uncontrolled trafficking of T-cells is implicated in many pathological conditions, including autoimmune disorders, such as psoriasis and inflammatory bowel disease. T-cell migration is regulated mainly by the αLβ2 integrin receptor LFA-1, which interacts primarily with its cognate ligand ICAM-1 expressed on the endothelium. This interaction triggers a plethora of downstream signaling pathways, which are not fully understood. Thus, in order to dissect ...
Source: Mol Biol Cell - August 21, 2019 Category: Molecular Biology Authors: Verma NK, Palapetta SM, Ong ST, Fazil MHUT, Chalasani MLS, Prasannan P, Kizhakeyil A, Kelleher D Tags: Methods Mol Biol Source Type: research

Quantification of T-Cell Migratory Phenotypes Using High-Content Analysis.
Abstract The exploration screening of phenotypic changes in motile T-cells within a signaling environment has always been an arduous task due to the sheer population of these microscopic cells. In recent years, High-Content Analysis (HCA) has gained epochal momentum and has allowed for a wider range of quantitative multiplexed cell-based assays in the field of lymphocyte signaling. In this chapter, we consolidate our understanding and describe the technical approach and methodology to quantify T-cell migratory phenotypes using HCA. Optimizations to be adopted to generate high-quality cytological images of motile T...
Source: Mol Biol Cell - August 21, 2019 Category: Molecular Biology Authors: Ng AS, Ong ST, Kelleher D, Verma NK Tags: Methods Mol Biol Source Type: research

Live Cell Imaging and Analysis to Capture T-Cell Motility in Real-Time.
Abstract T-lymphocytes are the principle coordinators of the immune defense system and play a major role in the protection of our body against infections, intruders of non-self, and malignancies. To mount an immune response, T-cells need to be effectively employed to tissue sites of infection or inflammation and establish contacts with antigen-presenting cells (APCs) or malignant cells. Understanding how T-cells navigate toward their recruitment sites would offer new therapeutic opportunities. Advancement in the hardware and software upgrades of microscopy technology has created several efficient and easy-to-opera...
Source: Mol Biol Cell - August 21, 2019 Category: Molecular Biology Authors: Ong ST, Verma NK Tags: Methods Mol Biol Source Type: research

Three-Dimensional Structured Illumination Microscopy (3D-SIM) to Dissect Signaling Cross-Talks in Motile T-Cells.
Abstract Visualization of signal transduction events in T-cells has always been a challenge due to their miniscule size. Recent advancement in super-resolution microscopy techniques presents many new opportunities to navigate the spatial and temporal signaling cross-talks in motile T-cells. Here, we provide technical details, optimal conditions, and critical practical considerations that need to be taken into account during cell handling, sample preparation, and image acquisition of motile T-cells for performing three-dimensional structured illumination microscopy (3D-SIM). PMID: 30610597 [PubMed - indexed fo...
Source: Mol Biol Cell - August 21, 2019 Category: Molecular Biology Authors: Ong ST, Wright GD, Verma NK Tags: Methods Mol Biol Source Type: research

Real-Time Impedance-Based Detection of LFA-1-Stimulated T-Cell Transwell Chemotaxis.
Abstract The ability of activated T-lymphocytes to transmigrate toward certain chemokine is one of their characteristic functional properties. Here, we provide step-wise details about an in vitro technique to quantify the kinetics of chemotactic behavior of LFA-1-stimulated T-lymphocytes. The method described herein utilizes a noninvasive electrical impedance-based detection system to monitor T-cell chemotaxis in real-time. PMID: 30610598 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - August 21, 2019 Category: Molecular Biology Authors: Prasannan P, Verma NK Tags: Methods Mol Biol Source Type: research

GapmeR-Mediated Gene Silencing in Motile T-Cells.
Abstract Gene silencing is an important method to study gene functions in health and diseases. While there are various techniques that are applied to knockdown specific gene(s) of interest, they have certain limitations in application to T-lymphocytes. T-cells are "hard-to-transfect" cells and are recalcitrant to transfection reagents. Here, we describe the use of novel cell-permeating antisense molecules, called "GapmeR", to knockdown specific gene(s) in human primary T-cells. PMID: 30610600 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - August 21, 2019 Category: Molecular Biology Authors: Fazil MHUT, Ong ST, Chalasani MLS, Kizhakeyil A, Verma NK Tags: Methods Mol Biol Source Type: research

Live Imaging of Resident T-Cell Migration in Human Lymphoid Tissue Slices Using Confocal Microscopy.
Abstract In order to mount a potent immune response, immune cells must move actively through tissues. As an example, T-cell need to migrate within lymph nodes in order to scan the surface of many dendritic cells and recognize rare expressed antigens. The recent development of improved imaging approaches, such as two-photon microscopy, and the use of powerful mouse models have shed light on some of the mechanisms that regulate the migration of immune cells in many organs. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly com...
Source: Mol Biol Cell - August 21, 2019 Category: Molecular Biology Authors: Donnadieu E, Michel Y, Hansmann ML Tags: Methods Mol Biol Source Type: research

Phosphoprotein Enrichment for Protein Analysis in Motile T-Lymphocytes.
Abstract Protein phosphorylation plays a key role in intracellular signal transduction and regulates diverse cellular functions. This posttranslational modification of proteins occurs dynamically and reversibly and only a small fraction of the total proteins is phosphorylated at any given time depending on the cell types and their functioning. Thus, a relatively low abundance of phosphorylated proteins is present in specific cells under certain conditions and hence it becomes problematic to detect these proteins and their analysis. In particular, phosphoproteomic analysis of rapidly migrating T-lymphocytes is alwa...
Source: Mol Biol Cell - August 21, 2019 Category: Molecular Biology Authors: Prasannan P, Saravanan R, Verma NK Tags: Methods Mol Biol Source Type: research

Immunometabolomic Phenotyping of Motile T-Cells.
Abstract The immune system and its components defend our body against diverse pathogens and help in maintaining tissue homeostasis. Immune cells are highly dynamic in terms of their growth, migration, differentiation, and effector functions, and adopt diverse metabolic configurations to respond to varying immunological challenges. Growing body of evidence suggests that metabolic pathways fuel immune cells for their functioning, including T-cell migration to the site of infection. This chapter provides detailed methodology for the efficient extraction of T-cell metabolites for successful downstream immunometabolomi...
Source: Mol Biol Cell - August 21, 2019 Category: Molecular Biology Authors: Chalasani MLS, Dong GLW, Verma NK Tags: Methods Mol Biol Source Type: research

Profiling Activity of Cellular Kinases in Migrating T-Cells.
Abstract T-Lymphocyte kinases are important checkpoints that control T-cell motility by regulating a diverse range of signal transduction pathways. The distinct configuration of kinase events in T-cell could be used to fingerprint the status of T-cells. However, only small fraction human kinases have been characterized so far and little is known about the dynamics of the kinome in motile T-cells. Although several direct and indirect strategies exist to characterize cellular kinase activities, such as RNA interference, antibody arrays, enzyme kinetics, and mass spectrometry, this chapter focuses on an alternative m...
Source: Mol Biol Cell - August 21, 2019 Category: Molecular Biology Authors: Chirumamilla CS, Fazil MHUT, Perez-Novo C, Rangarajan S, de Wijn R, Ramireddy P, Verma NK, Vanden Berghe W Tags: Methods Mol Biol Source Type: research