Sandwich-Based Antibody Arrays for Protein Detection.
Abstract Sandwich-based antibody arrays enable the detection of multiple proteins simultaneously, thus offering a time- and cost-effective alternative to single-plex platforms. The protein of interest is "sandwiched" between an antibody that captures it to the array and a second antibody that is used for detection. Here we describe a 1-day procedure to process samples, such as serum or cell lysates, with a quantitative sandwich-based antibody array on a glass substrate using fluorescence. PMID: 33237404 [PubMed - as supplied by publisher] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 26, 2020 Category: Molecular Biology Authors: Mao YQ, Petritis B, Huang RP Tags: Methods Mol Biol Source Type: research

Antibody Arrays: Barcode Technology.
Abstract Antibody microarray is a fundamental, high-content technology for analyzing biomarkers with a multiplexity even at the proteomic level. Recent advancement in this field has driven the antibody array into a new territory related with single-cell analysis. Here we describe a flow pattern-based method for producing a high-density barcode antibody microarray for the detection of proteins in fluidic samples and in single cells. The antibody microarray is fabricated by a perpendicularly oriented flow patterning of single-stranded barcode DNAs, which are then converted into DNA-antibody conjugates. Compared to c...
Source: Mol Biol Cell - November 26, 2020 Category: Molecular Biology Authors: Yang L, Wang J Tags: Methods Mol Biol Source Type: research

Profiling Glycoproteins on Functionalized Reverse Phase Protein Array.
Abstract Reverse phase protein array (RPPA), a high-throughput, parallel immunoassay in a dot-blot format, is a powerful tool to quantitatively profile protein expression in multiple samples simultaneously using small amounts of material. Despite its success, analysis of post-translationally modified (PTM) proteins has been limited in RPPA assays, primarily due to relatively low availability of antibodies specific to proteins of PTMs, e.g., glycosylation. Moreover, the high matrix complexity, with tens of thousands of proteins in cell lysates or tissue extracts and the low abundance of proteins with PTMs, makes it...
Source: Mol Biol Cell - November 26, 2020 Category: Molecular Biology Authors: Zhang Y, Zhang L, Iliuk A, Tao WA Tags: Methods Mol Biol Source Type: research

Determining Protein Phosphorylation Status Using Antibody Arrays and Phos-Tag Biotin.
We describe here a standard protocol for determining the phosphorylation status of protein multiplexes using antibody arrays and a biotinylated Phos-tag with a dodeca(ethylene glycol) spacer (Phos-tag Biotin). The procedure is based on an antibody microarray technique used in conjunction with an enhanced chemiluminescence system, and it permits the simultaneous and highly sensitive detection of multiple phosphoproteins in a cell lysate. By using this procedure, we have demonstrated the quantitative detection of the entire phosphorylation status of a target protein involved in intracellular signaling. PMID: 33237421 [P...
Source: Mol Biol Cell - November 26, 2020 Category: Molecular Biology Authors: Kinoshita E, Kinoshita-Kikuta E, Koike T Tags: Methods Mol Biol Source Type: research

Analyzing Signaling Pathways Using Antibody Arrays.
Abstract Cell signaling is comprised of complex networks that regulate homeostasis and human diseases. The analyses of such pathways would improve our understanding of disease pathology and direct drug development. However, it remains a great challenge to study pathways using traditional methods. We developed a high-throughput sandwich-based antibody array technology for the simultaneous detection of multiple targets, capable of identifying the relative expression levels or phosphorylation levels of major signaling pathway proteins. This array-based system features a nitrocellulose membrane or glass slide solid su...
Source: Mol Biol Cell - November 26, 2020 Category: Molecular Biology Authors: Tang H, Duan C, Kuang Z, Huang RP Tags: Methods Mol Biol Source Type: research

Immune Cell Isolation from Murine Intestine for Antibody Array Analysis.
Abstract Gut mucosal immune cells play an essential role in health due to their ability to orchestrate host signaling events in response to exogenous antigens. These antigens may originate from microorganisms including viruses, commensal or pathogenic bacteria, or single-celled eukaryotes, as well as from dietary foodstuff-derived proteins or products. A critical technological capacity to understand host responses to antigens is the ability to efficiently isolate and functionally characterize immune cells from intestinal tissues. Additionally, after characterization, it is of paramount importance to understand the...
Source: Mol Biol Cell - November 26, 2020 Category: Molecular Biology Authors: Owens JA, Jones RM Tags: Methods Mol Biol Source Type: research

Immunophenotyping of Circulating Myeloid-Derived Suppressor Cells (MDSC) in the Peripheral Blood of Cancer Patients.
Abstract Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of pathologically expanded myeloid cells with immunosuppressive activity. According to their phenotype, MDSC can be divided into three major subpopulations: early stage MDSC (e-MDSC), lacking myeloid lineage markers, monocytic MDSC (M-MDSC), and granulocytic MDSC (PMN-MDSC). Additionally, PMN-MDSC can be subdivided based on their activation and differentiation status, although it is not clear how this status contributes to immunosuppression and disease pathology. Here, we describe an immunophenotyping and gating strategy for the identificat...
Source: Mol Biol Cell - November 26, 2020 Category: Molecular Biology Authors: Bruderek K, Schirrmann R, Brandau S Tags: Methods Mol Biol Source Type: research

Isolation and Phenotyping of Splenic Myeloid-Derived Suppressor Cells in Murine Cancer Models.
Abstract Myeloid-derived suppressor cells (MDSC) are immunosuppressive myeloid cells that accumulate in tumor sites and peripheral lymphoid organs such as the spleen. In murine cancer models, the spleen is a major reservoir for MDSC, representing an easily accessible tissue from which to isolate high numbers of these cell population for downstream applications. Here we describe an efficient method to phenotype as well as to isolate and assess the functionality of murine splenic MDSC. PMID: 33237537 [PubMed - as supplied by publisher] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 26, 2020 Category: Molecular Biology Authors: Sanseviero E, Kim R, Gabrilovich DI Tags: Methods Mol Biol Source Type: research

Phenotypical Characterization and Isolation of Tumor-Derived Mouse Myeloid-Derived Suppressor Cells.
ve; S Abstract Myeloid-derived suppressor cells (MDSCs) are a heterogeneous cell population composed of mature and immature cells of myeloid origin that play a major role in tumor progression by inhibiting the antitumor immune responses mediated by T cells. In this chapter, we describe protocols for isolation, phenotypical and functional evaluation of MDSCs isolated from mouse tumors, with the aim at unifying and standardizing protocols set up by different laboratories. PMID: 33237538 [PubMed - as supplied by publisher] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 26, 2020 Category: Molecular Biology Authors: Barouni RM, Musiu C, Bronte V, Ugel S, Canè S Tags: Methods Mol Biol Source Type: research

Isolation of Human Circulating Myeloid-Derived Suppressor Cells and Analysis of Their Immunosuppressive Activity.
Abstract Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of myeloid cells with potent immunosuppressive activity and characterized by a pathological state of activation. The T cell suppression assay is the most common method to evaluate the suppressive capacity of MDSC. Identifying the suppressive potential of different MDSC subsets within individual donors is key for understanding the biology of MDSC and their clinical relevance. Here we describe assays to ascertain and quantify the suppression of autologous T cells by human MDSC. These include the dye dilution proliferation assay for flow ...
Source: Mol Biol Cell - November 26, 2020 Category: Molecular Biology Authors: Bruderek K, Schirrmann R, Brandau S Tags: Methods Mol Biol Source Type: research

In Vitro Generation of Human Neutrophilic Myeloid-Derived Suppressor Cells.
Abstract Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells of myeloid origin. MDSC are functionally defined by their capacity to suppress T, NK, and B cell responses and henceforth altering the disease outcome in various pathological conditions. MDSC are further subdivided into three distinct subsets: monocytic (M-) MDSC, neutrophilic or polymorphonuclear (PMN-) MDSC, and early-stage (e-) MDSC. However, since surface markers utilized to define MDSC are expressed on other myeloid cells too, it is mandatory to functionally assess the suppressive activity for characterizing these cells. ...
Source: Mol Biol Cell - November 26, 2020 Category: Molecular Biology Authors: Singh A, Rieber N Tags: Methods Mol Biol Source Type: research

Measuring Suppressive Activity and Autophagy in Myeloid-Derived Suppressor Cells.
Abstract Myeloid-derived suppressor cells (MDSC) are potent suppressor cells that accumulate in tumor microenvironment and inhibit anti-tumor responses. Assessment of cell-autonomous MDSC responses allows the precise characterization of MDSCs in various disease settings and elucidates the underlying mechanisms of MDSC-mediated immune suppression. Here we describe a protocol for the isolation of tumor infiltrating or splenic MDSC, as well as their subpopulations, from melanoma-inoculated mice using Fluorescent Activated Cell Sorting (FACS). We further provide protocols for investigation of the autophagy pathway and...
Source: Mol Biol Cell - November 26, 2020 Category: Molecular Biology Authors: Papaioannou AS, Boumpas A, Papadopoulou M, Hatzioannou A, Alissafi T, Verginis P Tags: Methods Mol Biol Source Type: research

Analysis of Antimicrobial Activity of Monocytic Myeloid-Derived Suppressor Cells in Infection with Mycobacterium tuberculosis and Human Immunodeficiency Virus.
Abstract Myeloid-derived suppressor cells (MDSC) encompass a subset of myeloid cells, which suppress both innate and adaptive immune functions. Since Mycobacterium tuberculosis (M. tuberculosis) can infect these cells, interest has emerged to study the antimicrobial response of MDSC to mycobacteria causing tuberculosis. Reactive oxygen species (ROS) are critical mediators to control intracellular replication of M. tuberculosis and MDSC express high levels of these effector molecules. Here we describe the flow cytometric assessment of total cellular ROS produced by MDSC in response to infection with M. tuberculosis...
Source: Mol Biol Cell - November 26, 2020 Category: Molecular Biology Authors: Garg A Tags: Methods Mol Biol Source Type: research

Isolation and Functional Characterization of Myeloid-Derived Suppressor Cells in Infections Under High Containment.
Abstract The current absence of markers unique to MDSC, particularly those expanded during human infection, necessitate concurrent demonstration of their suppressive capacity to ensure unequivocal identification. This is further complicated by the array of heterogeneous markers used to characterize MDSC in various conditions and models. Standardization of phenotypic and functional characterization, as well as isolation, from infectious biological samples of patients, are critical for accurately reporting MDSC dynamics, function, organ abundance, and establishment of their therapeutic value in infectious diseases. ...
Source: Mol Biol Cell - November 26, 2020 Category: Molecular Biology Authors: Kotze LA, Leukes VN, Du Plessis N Tags: Methods Mol Biol Source Type: research

High-Dimensional Analysis of Circulating and Tissue-Derived Myeloid-Derived Suppressor Cells from Patients with Glioblastoma.
Abstract We will first describe analysis of MDSC subsets from patient tumors with multicolor flow cytometry. The key components of this methodology are to obtain viable single cell suspensions and eliminate red blood cell contamination. PMID: 33237547 [PubMed - as supplied by publisher] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 26, 2020 Category: Molecular Biology Authors: Alban TJ, Bayik D, Alvarado AG, Kornblum HI, Lathia JD Tags: Methods Mol Biol Source Type: research

Single-Cell Transcriptome Analysis Workflow for Splenic Myeloid-Derived Suppressor Cells from Murine Breast Cancer Models.
Abstract Single-cell transcriptomics is a powerful tool to study previously unrealized cellular heterogeneity at the resolution of individual cells. Most of the previous knowledge in cell biology is based on data generated by bulk analysis methods, which provide averaged readouts that usually mask cellular heterogeneity. This approach is challenging when the biological effect of interest is limited to a subpopulation within a cell type. This may particularly apply immune cell populations as these cells are highly mobile and swiftly respond to changes in cytokines or chemokines. For example, in cancer certain subse...
Source: Mol Biol Cell - November 26, 2020 Category: Molecular Biology Authors: Alshetaiwi H, Pervolarakis N, Nguyen QH, Kessenbrock K Tags: Methods Mol Biol Source Type: research

Intravital 2-Photon Microscopy of Diverse Cell Types in the Murine Tibia.
Abstract Intravital imaging allows the visualization of fluorescently labeled structures like cells, blood flow, and pathogens in a living organism. Nowadays, numerous methods for imaging in several organs are available. In this chapter, we present a method for intravital 2-photon microscopy of the murine tibial bone marrow. It enables the observation of hematopoietic cells including cells of the innate and adaptive immune system under physiological conditions. Motility analyses within this complex environment led to insights into their migratory potential as well as their interactions with other cells or blood ve...
Source: Mol Biol Cell - November 26, 2020 Category: Molecular Biology Authors: Hasenberg A, Otto L, Gunzer M Tags: Methods Mol Biol Source Type: research

Isolation of Bovine Neutrophils by Fluorescence- and Magnetic-Activated Cell Sorting.
Abstract Flow cytometry and magnetic bead technology enable the separation of cell populations with the highest degree of purity. Here, we describe protocols to sort bovine neutrophils from blood, the labeling and sorting, including gating strategies. We also provide advice to preserve neutrophil viability and detail a protocol to measure phagocytosis and oxidative species production. PMID: 33237550 [PubMed - as supplied by publisher] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 26, 2020 Category: Molecular Biology Authors: Rambault M, Borkute R, Doz-Deblauwe E, Le-Vern Y, Winter N, Dorhoi A, Remot A Tags: Methods Mol Biol Source Type: research

Assessment of Immune Responses in an Animal Model of Wheat Food Allergy via Epicutaneous Sensitization.
Abstract Wheat allergy is a pathological event involving immunocompetent cells against ingested wheat allergen and is clearly associated with transdermal sensitization. However, the molecular mechanisms involved in the disease etiology are not completely understood. A complex cellular and tissue network linking to food allergy makes it difficult to understand the molecular mechanism of allergenicity. Animal models are valuable tools to deduce basic principles of human disease without invasive intervention trials. A mouse model of wheat allergy has provided insights into effects of skin exposure to wheat protein; i...
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Tamehiro N, Adachi R, Kondo K Tags: Methods Mol Biol Source Type: research

A Mouse Model of Oral Sensitization to Hen's Egg White.
lde;o R, Molina E Abstract Egg allergy is one of the most common food allergies in children, being the most important allergenic proteins found in the egg white (EW). Allergy to EW shows a complex phenotype that involves a multifaceted reaction that can only be assessed in vivo. Although other routes of sensitization have been described, oral exposure to food antigens is one of the most suitable in humans. In mice, oral administration of allergenic proteins results in the development of tolerance, and the use of adjuvants, such as cholera toxin (CT), is required to promote Th2-biased immune responses over toleroge...
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Benedé S, Lozano-Ojalvo D, López-Fandiño R, Molina E Tags: Methods Mol Biol Source Type: research

Animal Models of Contact Dermatitis: 2,4-Dinitrofluorobenzene-Induced Contact Hypersensitivity.
Abstract Allergic contact dermatitis (ACD) is a common skin disease with high prevalence in work environments. Human allergic contact dermatitis is triggered by the exposure to haptens that leads to an initial phase known as sensitization. During this phase, hapten-protein complexes presented by antigen-presenting cells activate a T-cell-mediated response, leading to the generation of memory cells against the hapten. Upon re-exposure to the same hapten, the elicitation phase is initiated. This phase is characterized by a quicker acute inflammatory response involving activation and/or infiltration of a variety of i...
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Manresa MC Tags: Methods Mol Biol Source Type: research

Experimental Mouse Models of Ragweed- and Papain-Induced Allergic Conjunctivitis.
Abstract Mouse models of allergic conjunctivitis mimic various aspects of human allergic conjunctivitis. They are useful as acute models of allergic conjunctivitis to study immunological aspects of this condition. In this chapter, we will describe ragweed-pollen-induced experimental allergic conjunctivitis (mostly driven by adaptive immunity), and papain-soaked contact lens-induced experimental allergic conjunctivitis (mostly driven by innate immunity). Giemsa staining of histological sections is used for quantification of the number of infiltrating eosinophils, which is useful to evaluate the severity of the alle...
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Matsuda A, Hirakata T, Asada Y, Nakae S Tags: Methods Mol Biol Source Type: research

An Overview of Flow Cytometry: Its Principles and Applications in Allergic Disease Research.
Abstract Flow cytometry is a popular technique used for both clinical and research purposes. It involves laser-based technology to characterize cells based on size, shape, and complexity. Additionally, flow cytometers are equipped with the ability to take fluorescence measurements at multiple wavelengths. This capability makes the flow cytometer a practical resource in the utilization of fluorescently conjugated antibodies, fluorescent proteins, DNA binding dyes, viability dyes, and ion indicator dyes. As the technology advances, the number of parameters a flow cytometer can measure has increased tremendously, and...
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Schmit T, Klomp M, Khan MN Tags: Methods Mol Biol Source Type: research

The Application of Flow Cytometry for Simultaneous and Multi-parametric Analysis of Heterogenous Cell Populations in Basic and Clinical Research.
Abstract The use of flow cytometry allows simultaneous measurement and multiparametric analysis of single cells in a heterogenous solution. The purpose of flow cytometry can vary depending on the use of antibodies and dyes targeted for specific cell molecules. The method of immune-phenotyping with fluorescently conjugated antibodies to label cell proteins or DNA works in tandem with fluidic, optic, and electrical systems present in the flow cytometer. Some flow cytometers can detect numerous fluorescent molecules on a single cell, allowing the measurement of more than 30 parameters. This ability to detect, measure...
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Schmit T, Klomp M, Khan MN Tags: Methods Mol Biol Source Type: research

Preservation and Processing of Intestinal Tissue for the Assessment of Histopathology.
We describe the manual processing that is often used for smaller batches of samples. PMID: 33226600 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Rieger J, Pelckmann LM, Drewes B Tags: Methods Mol Biol Source Type: research

Methods for Experimental Allergen Immunotherapy: Subcutaneous and Sublingual Desensitization in Mouse Models of Allergic Asthma.
Abstract Allergic asthma is characterized by airway hyperresponsiveness, remodeling, and reversible airway obstruction. This is associated with an eosinophilic inflammation of the airways, caused by inhaled allergens such as house dust mite or grass pollen. The inhaled allergens trigger a type-2 inflammatory response with the involvement of innate lymphoid cells (ILC2) and Th2 cells, resulting in high immunoglobulin E (IgE) antibody production by B cells and mucus production by airway epithelial cells. As a consequence of the IgE production, subsequent allergen reexposure results in a classic allergic response wit...
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Hesse L, Petersen AH, Nawijn MC Tags: Methods Mol Biol Source Type: research

T-Cell Epitope Immunotherapy in Mouse Models of Food Allergy.
Abstract Food allergy has been rising in prevalence over the last two decades, affecting more than 10% of the world population. Current management of IgE-mediated food allergy relies on avoidance and rescue medications; research into treatments that are safer and providing guaranteed and durable curative effects is, therefore, essential. T-cell epitope-based immunotherapy holds the potential for modulating food allergic responses without IgE cross-linking. In this chapter, we describe the methods in evaluating the therapeutic capacities of immunodominant T-cell epitopes in animal models of food allergy. Moreover, ...
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Wai CYY, Leung NYH, Chu KH, Leung PSC Tags: Methods Mol Biol Source Type: research

Pig Cloning Using Somatic Cell Nuclear Transfer.
Abstract Porcine cloning technology can be used to produce progenies genetically identical to the donor cells from high-quality breeding pigs. In addition, genetically modified pigs have been produced by somatic cell nuclear transfer using genetically modified porcine fetal fibroblasts. The method of preparing genetically modified pigs is critical for establishing pig models for human diseases, and for generating donor animals for future xenotransplantation. This chapter describes detailed procedures for generating cloned pigs using fetal fibroblasts as nuclear donors. PMID: 33226609 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Ouyang H, Han J, Huang Y Tags: Methods Mol Biol Source Type: research

Cloning of Monkeys by Somatic Cell Nuclear Transfer.
Abstract Somatic cell nuclear transfer (SCNT) is a promising method to establish genetically modified monkeys with identical genetic background as models in biomedical research. We have recently cloned monkeys by optimization of the SCNT protocols and inclusion of the epigenetic modulator. Here, we describe the protocol for generation of cloned monkeys by somatic cell nuclear transfer. PMID: 33226610 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Sun S, Liao Z, Liu Z, Sun Q Tags: Methods Mol Biol Source Type: research

Production of Cardiomyocyte-Like Cells by Fibroblast Reprogramming with Defined Factors.
Abstract Over the last decade, great achievements have been made in the field of direct epigenetic reprogramming, which converts one type of adult somatic cells into another type of differentiated cells, such as direct reprogramming of fibroblasts into cardiomyocytes, without passage through an undifferentiated pluripotent stage. Discovery of direct cardiac reprogramming offers a promising therapeutic strategy to prevent/attenuate cardiac fibrotic remodeling in a diseased heart. Furthermore, in vitro reprogramming of fibroblasts into cardiomyocyte-like cells provides new avenues to conduct basic mechanistic studie...
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Bektik E, Fu JD Tags: Methods Mol Biol Source Type: research

Direct Reprogramming of Human Fibroblasts into Induced Neural Progenitor Cells Using Suicide Gene Embodied Episomal Vectors for Rapid Selection of Exogenous DNA-Free Cells.
Abstract Direct neural reprogramming involves a rapid conversion of somatic cells into neural cells without passing through the intermediate pluripotent stage. This phenomenon can be mediated in the starting somatic cells by the introduction of lineage-specific master transcription factors or by pluripotency factors routinely used in iPS cell generation. In the latter process known as Pluripotency factor-mediated Direct Reprogramming (PDR), the pluripotency factors are used to elicit epigenetic changes producing a permissive state in the starting cells which are then driven to the neural lineages by simple manipul...
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Lee M, Kim J, Ambasudhan R Tags: Methods Mol Biol Source Type: research

Generation of Human Neurons by microRNA-Mediated Direct Conversion of Dermal Fibroblasts.
Abstract MicroRNAs (miRNAs), miR-9/9*, and miR-124 (miR-9/9*-124) display fate-reprogramming activities when ectopically expressed in human fibroblasts by erasing the fibroblast identity and evoking a pan-neuronal state. In contrast to induced pluripotent stem cell-derived neurons, miRNA-induced neurons (miNs) retain the biological age of the starting fibroblasts through direct fate conversion and thus provide a human neuron-based platform to study cellular properties inherent in aged neurons and model adult-onset neurodegenerative disorders using patient-derived cells. Furthermore, expression of neuronal subtype-...
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Church VA, Cates K, Capano L, Aryal S, Kim WK, Yoo AS Tags: Methods Mol Biol Source Type: research

Episomal Reprogramming of Human Peripheral Blood Mononuclear Cells into Pluripotency.
Abstract Peripheral blood is an easily accessible cell resource for reprogramming into pluripotency by episomal vectors. Here, we describe an approach for efficient generation of integration-free induced pluripotent stem cells (iPSCs) under feeder or feeder-free conditions. Additionally, in combination with the CRISPR-Cas9 genome-editing system, we can directly generate edited iPSCs from blood cells. With this protocol, one can easily generate either integration-free iPSCs or genetically edited iPSCs from peripheral blood at high efficiency. PMID: 33226616 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Wen W, Cheng T, Zhang XB Tags: Methods Mol Biol Source Type: research

Generation of Human iPSCs by Episomal Reprogramming of Skin Fibroblasts and Peripheral Blood Mononuclear Cells.
Abstract Human-induced pluripotent stem cells (iPSCs) can be generated from patient-specific somatic cells by forced expression of the transcription factors OCT4, SOX2, KLF4, and c-MYC. Sustained expression of the transgenes during reprogramming is crucial for the successful derivation of iPSCs. Integrating retroviruses have been used to achieve the required prolonged expression; however, issues of undesirable transgene expression in the iPSC-derived cell types post reprogramming can occur. Alternative non-integrating approaches to reprogram somatic cells into pluripotency have been established. Here, we describe ...
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Febbraro F, Chen M, Denham M Tags: Methods Mol Biol Source Type: research

Generation of Human iPSCs by Protein Reprogramming and Stimulation of TLR3 Signaling.
Abstract The discovery of induced pluripotent stem cells (iPSCs) allows for establishment of human embryonic stem-like cells from various adult human somatic cells (e.g., fibroblasts), without the need for destruction of human embryos. This provides an unprecedented opportunity where patient-specific iPSCs can be subsequently differentiated to many cell types, e.g., cardiac cells and neurons, so that we can use these iPSC-derived cells to study patient-specific disease mechanisms and conduct drug testing and screening. Critically, these cells have unlimited therapeutic potentials, and there are many ongoing clinic...
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Liu C, Ameen M, Himmati S, Thomas D, Sayed N Tags: Methods Mol Biol Source Type: research

Reprogramming of Fibroblasts to Human iPSCs by CRISPR Activators.
Abstract CRISPR-mediated gene activation (CRISPRa) can be used to target endogenous genes for activation. By targeting pluripotency-associated reprogramming factors, human fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSCs). Here, we describe a method for the derivation of iPSCs from human fibroblasts using episomal plasmids encoding CRISPRa components. This chapter also provides procedure to assemble guide RNA cassettes and generation of multiplexed guide plasmids for readers who want to design their own guide RNAs. PMID: 33226620 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Weltner J, Trokovic R Tags: Methods Mol Biol Source Type: research

Reprogramming Porcine Fibroblast to EPSCs.
Abstract The development of porcine expanded potential stem cells (pEPSCs) provides an invaluable tool for investigation of porcine stem cell pluripotency and opens a venue for research in biotechnology, agriculture, and regenerative medicine. Since the derivation of pEPSC from porcine pre-implantation embryos has been demanding in resource supply and technical challenges, it is more feasible and convenient for most laboratories to derive this new type of porcine stem cells by reprogramming somatic cells. In this chapter, we describe the detailed procedures for reprogramming porcine fetal fibroblast cells to EPSCi...
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Gao X, Ruan D, Liu P Tags: Methods Mol Biol Source Type: research

Evaluating Reprogramming Efficiency and Pluripotency of the Established Human iPSCS by Pluripotency Markers.
Abstract The pluripotency of human induced pluripotent stem cells (HiPSCs) cannot be tested strictly in a similar way as we can do for the mouse ones because of ethical restrictions. One common and initial approach to prove the pluripotency of an established human iPSC line is to demonstrate expression of a set of established surface and intracellular pluripotency markers. This chapter provides procedures of immunocytochemistry of the established HiPSC lines for a set of the signature intracellular pluripotency proteins, OCT4, SOX2, NANOG, and LIN28. We also describe cell phenotyping by flow cytometry for the five...
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Cevallos RR, Hossain ME, Zhang R, Hu K Tags: Methods Mol Biol Source Type: research

G-Banded Karyotyping of Human Pluripotent Stem Cell Cultures.
Abstract Acquired chromosomal abnormalities may occur during the reprogramming and culture of human pluripotent stem cells (hPSCs). Therefore, it is required that regular testing of genetic integrity be conducted. G-banded karyotyping is a widely used genetic assay that is capable of detecting chromosomal abnormalities. Karyotyping of hPSC cultures can be a challenging undertaking for inexperienced investigators; here, we provide detailed procedures for karyotyping, including sample preparation and analysis, as well as the interpretation of hPSC karyotype results. PMID: 33226624 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: McIntire E, Leonhard K, Taapken S, Larson AL Tags: Methods Mol Biol Source Type: research

In Vitro Analysis of mtDNA Replication.
Abstract Human mitochondrial DNA is a small circular double-stranded molecule that is essential for cellular energy production. A specialized protein machinery replicates the mitochondrial genome, with DNA polymerase γ carrying out synthesis of both strands. According to the prevailing mitochondrial DNA replication model, the two strands are replicated asynchronously, with the leading heavy-strand initiating first, followed by the lagging light-strand. By using purified recombinant forms of the replication proteins and synthetic DNA templates, it is possible to reconstitute mitochondrial DNA replication in v...
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Uhler JP, Falkenberg M Tags: Methods Mol Biol Source Type: research

Visualization of Mitochondrial RNA Granules in Cultured Cells Using 5-Bromouridine Labeling.
Abstract The incorporation of nucleoside analogs is a useful tool to study the various functions of DNA and RNA. These analogs can be detected directly by fluorescence or by immunolabeling, allowing to visualize, track, or measure the nucleic acid molecules in which they have been incorporated. In this chapter, methodologies to measure human mitochondrial transcription are described. The nascent RNA that is transcribed from mitochondrial DNA (mtDNA) has been shown to assemble into large ribonucleoprotein complexes that form discrete foci. These structures were called mitochondrial RNA granules (MRGs) and can be ob...
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Xavier VJ, Martinou JC Tags: Methods Mol Biol Source Type: research

RNA Crosslinking to Analyze the Mitochondrial RNA-Binding Proteome.
Abstract Even though the mammalian mitochondrial genome (mtDNA) is very small and only codes for 13 proteins, all being subunits of the oxidative phosphorylation system, it requires several hundred nuclear encoded proteins for its maintenance and expression. These include replication and transcription factors, approximately 80 mitoribosomal proteins and many proteins involved in the posttranscriptional modification, processing, and stability of mitochondrial RNAs. In recent years, many of these factors have been identified and functionally characterized, but the complete mtRNA-interacting proteome is not firmly es...
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: van Esveld SL, Spelbrink JN Tags: Methods Mol Biol Source Type: research

Visualizing Mitochondrial Ribosomal RNA and Mitochondrial Protein Synthesis in Human Cell Lines.
Abstract Human mitochondria contain their own DNA (mtDNA) that encodes 13 proteins all of which are core subunits of oxidative phosphorylation (OXPHOS) complexes. To form functional complexes, these 13 components need to be correctly assembled with approximately 70 nuclear-encoded subunits that are imported following synthesis in the cytosol. How this complicated coordinated translation and assembly is choreographed is still not clear. Methods are being developed to determine whether all members of a particular complex are translated in close proximity, whether protein synthesis is clustered in submitochondrial fa...
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Zorkau M, Proctor-Kent Y, Berlinguer-Palmini R, Hamilton A, Chrzanowska-Lightowlers ZM, Lightowlers RN Tags: Methods Mol Biol Source Type: research

Mitoribosome Profiling from Human Cell Culture: A High Resolution View of Mitochondrial Translation.
Abstract Ribosome profiling (Ribo-Seq) is a technique that allows genome-wide, quantitative analysis of translation. In recent years, it has found multiple applications in studies of translation in diverse organisms, tracking protein synthesis with single codon resolution. Traditional protocols applied for generating Ribo-Seq libraries from mammalian cell cultures are not suitable to study mitochondrial translation due to differences between eukaryotic cytosolic and mitochondrial ribosomes. Here, we present an adapted protocol enriching for mitoribosome footprints. In addition, we describe the preparation of small...
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Pearce SF, Cipullo M, Chung B, Brierley I, Rorbach J Tags: Methods Mol Biol Source Type: research

The Analysis of Yeast Mitochondrial Translation.
t M Abstract The mitochondrial genome encodes only a handful of proteins, but methods to track their synthesis are highly limited. Saccharomyces cerevisiae is a model organism that offers possibilities to expand the classical systems to analyze mitochondrial translation. In this chapter, we present two approaches of monitoring mitochondrial protein synthesis. Labeling of mitochondrially translated products with radioactive amino acids can be performed either in intact cells or in isolated mitochondria. However, these classical methods have disadvantages that can affect cell physiology and hence are not suitable fo...
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Carlström A, Rzepka M, Ott M Tags: Methods Mol Biol Source Type: research

Mitochondrial Complexome Profiling.
Abstract Complexome profiling combines blue native gel electrophoresis (BNE) and quantitative mass spectrometry to define an entire protein interactome of a cell, an organelle, or a biological membrane preparation. The method allows the identification of protein assemblies with low abundance and detects dynamic processes of protein complex assembly. Applications of complexome profiling range from the determination of complex subunit compositions, assembly of single protein complexes, and supercomplexes to comprehensive differential studies between patients or disease models. This chapter describes the workflow of ...
Source: Mol Biol Cell - November 25, 2020 Category: Molecular Biology Authors: Giese H, Meisterknecht J, Heidler J, Wittig I Tags: Methods Mol Biol Source Type: research

Stoichiometry of Receptors at the Plasma Membrane During Their Endocytosis Using Total Internal Reflection Fluorescent (TIRF) Microscopy Live Imaging and Single-Molecule Tracking.
Abstract Determination of protein stoichiometry in living cells is key to understanding basic biological processes. This is particularly important for receptor-mediated endocytosis, a highly regulated mechanism that requires the sequential assembly of numerous factors. Here, we describe a quantitative approach to analyze receptor clustering dynamics at the plasma membrane. Our workflow combines TIRF live imaging of a CRISPR-Cas9-edited cell line expressing a GFP-tagged receptor in a physiological relevant environment, a calibration technique for single-molecule analysis of GFP, and detection and tracking with...
Source: Mol Biol Cell - November 24, 2020 Category: Molecular Biology Authors: Salavessa L, Sauvonnet N Tags: Methods Mol Biol Source Type: research

Measuring Endocytosis During Proliferative Cell Quiescence.
Abstract Quiescence (also called "G0") is the state in which cells have exited the cell cycle but are capable to reenter as required. Though poorly understood, it represents one of the most prevalent cell states across all life. Many biologically important cell types reside in quiescence including mature hepatocytes, endothelial cells, and dormant adult stem cells. Furthermore, the quiescence program occurs in both short- and long-term varieties, depending on the physiological environments. A barrier slowing our understanding of quiescence has been a scarcity of available in vitro model systems to allow ...
Source: Mol Biol Cell - November 24, 2020 Category: Molecular Biology Authors: Hinze C, McGourty K, Boucrot E Tags: Methods Mol Biol Source Type: research

Measurements of Compensatory Endocytosis by Antibody Internalization and Quantification of Endocytic Vesicle Distribution in Adrenal Chromaffin Cells.
Abstract Plasma membrane proteins are amenable to endocytosis assays since they are easily labeled by reagents applied in the extracellular medium. This has been widely exploited to study constitutive endocytosis or ligand-induced receptor endocytosis. Compensatory endocytosis is the mechanism by which components of secretory vesicles are retrieved after vesicle fusion with the plasma membrane in response to cell stimulation and a rise in intracellular calcium. Luminal membrane proteins from secretory vesicles are therefore transiently exposed at the plasma membrane. Here, we described an antibody-based method to ...
Source: Mol Biol Cell - November 24, 2020 Category: Molecular Biology Authors: Ceridono M, Chasserot-Golaz S, Vitale N, Gasman S, Ory S Tags: Methods Mol Biol Source Type: research

Quantitative Methods to Study Endocytosis and Retrograde Transport of Cargo Proteins.
Abstract Endocytosis and intracellular retrograde trafficking from endosomes to the Golgi apparatus are key cellular processes. Endocytosis is directly or indirectly involved in many if not all cellular functions ranging from nutrient uptake and receptor signaling to mitosis, cell division, and migration (Scita, Di Fiore. Nature 463(7280):464-473, 2010; McMahon, Boucrot. Nat Rev Mol Cell Biol 12(8):517-533, 2011). Retrograde trafficking is emerging as a key driver for cell polarity. Robust methods are needed to quantify these processes. At the example of the bacterial Shiga toxin and the endogenous α5β1...
Source: Mol Biol Cell - November 24, 2020 Category: Molecular Biology Authors: Shafaq-Zadah M, Dransart E, Johannes L Tags: Methods Mol Biol Source Type: research