Isolation of Extracellular Vesicles from Leishmania spp.

Exosomes, a class of extracellular vesicles, are released by eukaryotes, bacteria, and archaea, as evident from both in vitro and in vivo studies. These nano-sized double-membraned vesicles play an important role in cell-to-cell communication, dysregulation of the immune system, and pathogenesis in a number of diseases, including leishmaniasis. Leishmania is a genus of obligate intracellular parasites, which infect host macrophages, are transmitted through the bite of a sandfly, and are shown to secrete exosomes with immunomodulatory activities. Given the importance of these vesicles in Leishmania spp. virulence, it is necessary to perform appropriate isolation and characterization in order to further study their relevance in the parasite's infectious life cycle. In this chapter, we describe four methods for the isolation of extracellular vesicles derived from Leishmania species including ultracentrifugation, polyethylene glycol-based precipitation, size-exclusion chromatography, and sucrose-gradient fractionation. Further, we describe the preparation of isolated samples for characterization by nanoparticle tracking analysis, transmission electron microscopy, and proteomic profiling.

A Scalable Purification Method for Mitochondria from Trypanosoma brucei.

Due to its unique biology the mitochondrion of Trypanosoma brucei has attracted a lot of interest since many decades, making it arguably the best studied mitochondrion outside yeast and mammals. Here we describe a method allowing purification of mitochondria from procyclic trypanosomes that yields highly enriched and functional organelles. The method is based on isotonic lysis of cells by nitrogen cavitation, DNase I digestion, differential centrifugation and Nycodenz gradient centrifugation. The method is scalable and can be adapted to culture volumes a small as 100 mL or as large as 24 L.

RNA Stable Isotope Probing (RNA-SIP).

Stable isotope probing is a combined molecular and isotopic technique used to probe the identity and function of uncultivated microorganisms within environmental samples. Employing stable isotopes of common elements such as carbon and nitrogen, RNA-SIP exploits an increase in the buoyant density of RNA caused by the active metabolism and incorporation of heavier mass isotopes into the RNA after cellular utilization of labeled substrates pulsed into the community. Labeled RNAs are subsequently separated from unlabeled RNAs by density gradient centrifugation followed by identification of the RNAs by sequencing. Therefore, RNA stable isotope probing is a culture-independent technique that provides simultaneous information about microbiome community, composition and function. This chapter presents the detailed protocol for performing an RNA-SIP experiment, including the formation, ultracentrifugation, and fractional analyses of stable isotope-labeled RNAs extracted from environmental samples.

R-DeeP: Proteome-wide and Quantitative Identification of RNA-Dependent Proteins by Density Gradient Ultracentrifugation.

The comprehensive but specific identification of RNA-binding proteins as well as the discovery of RNA-associated protein functions remain major challenges in RNA biology. Here we adapt the concept of RNA dependence, defining a protein as RNA dependent when its interactome depends on RNA. We converted this concept into a proteome-wide, unbiased, and enrichment-free screen called R-DeeP (RNA-dependent proteins), based on density gradient ultracentrifugation. Quantitative mass spectrometry identified 1,784 RNA-dependent proteins, including 537 lacking known links to RNA. Exploiting the quantitative nature of R-DeeP, proteins were classified as not, partially, or completely RNA dependent. R-DeeP identified the transcription factor CTCF as completely RNA dependent, and we uncovered that RNA is required for the CTCF-chromatin association. Additionally, R-DeeP allows reconstruction of protein complexes based on co-segregation. The whole dataset is available at http://R-DeeP.dkfz.de, providing proteome-wide, specific, and quantitative identification of proteins with RNA-dependent interactions and aiming at future functional discovery of RNA-protein complexes.

Two-dimensional planar swimming selects for high DNA integrity sperm.

Selection of high-quality sperm is critical to the success of assisted reproductive technologies. Clinical screening for top sperm has long focused on sperm swimming ability when following boundaries or when fully free of constraints. In this work, we demonstrate a sperm selection approach with parallel 2 µm tall confined selection channels that prohibit rotation of the sperm head and require planar swimming. We demonstrate that a planar swimming subpopulation of sperm capable of entering and navigating these channels has DNA integrity superior to the freely-swimming motile or raw sperm populations over a wide range of semen sample qualities. The DNA integrity of the selected sperm was significantly higher than that of the corresponding raw samples for donor samples and clinical patient samples, respectively. In side-by-side testing, this method outperforms current clinical selection methods, density gradient centrifugation and swim-up, as well as sperm selected via general motility. Planar swimming represents a viable sperm selection mechanism with the potential to improve outcomes for couples and offspring.

Immune Monitoring of Cancer Patients by Multi-color Flow Cytometry.

The irruption of immune-activating therapies to treat cancer has created a need for evaluating both the response and possible adverse events related to these novel treatments. Multicolor flow cytometry is a powerful tool that enables tumor immunologists to characterize the immune system of patients before and in response to immunotherapy. We present here a protocol for purifying human peripheral blood mononuclear cells and staining them with a set of six multicolor panels that allow for a thorough characterization of the immune system of healthy donors as well as patients that are undergoing treatments that may modify the immune system.

Measuring Nonselective and Selective Autophagy in the Liver.

Administration of leupeptin, a specific inhibitor of lysosomal cysteine proteinases, to starved rats or mice inhibits autolysosomal protein degradation and results in accumulation of autolysosomes in their livers. Immunoblotting of liver homogenates to examine autophagic flux in vivo reveals elevated levels of the selective autophagy substrate p62 and the autophagosomal membrane protein LC3-II in the livers of leupeptin-treated animals. Percoll density gradient centrifugation can be used to isolate autolysosomes from the livers of untreated and leupeptin-treated animals. Moreover, autolysosomes can be examined for the presence of sequestered cytoplasmic proteins as well as degradation intermediates.

Isolation of Extracellular Vesicles for Cancer Diagnosis and Functional Studies.

Extracellular vesicles (EVs) are a diverse category of cellular export products that are present in a variety of biofluids and cell culture media. EVs contain a wide variety of macromolecules that represent a sampling of the cytoplasmic or endosomal compartments and function in cell-to-cell paracrine and endocrine signaling; it has been demonstrated that pathological states such as oxidative stress, transformation, apoptosis, and various cell injuries induce cells to increase their EV release rate, simultaneously altering their composition to reflect the altered state of the cellular origin. Specifically, in patients with solid tumors, EVs are released from cancerous cells at a higher rate than from healthy cells and are enriched in tumor signature molecules. Because of their stability, increased concentration, and unique signatures in cancer patients, EVs have become the subject of investigation for diagnostic and prognostic purposes. Moreover, understanding EVs' biogenesis and biological role could lead to novel insights toward cellular cross talk and complex biological pathways in cancer research. To make use of EVs for diagnostic and mechanistic cancer research, standardized well-characterized methods are required. This chapter provides an overview of two EV isolation techniques and provides detailed instructions on the isolation of EVs by ultracentrifugation, the labor-intensive gold standard, and concentrated polymer precipitation, a faster, higher-yield technique that can be utilized in cancer research.

Ex Vivo Pharmacological Profiling in Chronic Lymphocytic Leukemia Cells.

In vitro drug combination studies are commonly used for CLL primary lymphocytes. An advancement in this method is to perform ex vivo drug testing where the first agent is administered to patients and second drug is tested in these patients' cells in vitro. These assays have been effective in identifying novel agents that work additively or synergistically. In this chapter, we provide a step-by-step protocol for ex vivo drug testing that can be used for combination strategies.

Mouse Model of Colitis-Associated Colorectal Cancer (CAC): Isolation and Characterization of Mucosal-Associated Lymphoid Cells.

Colorectal cancer (CRC) is the third most common malignancy worldwide presenting high mortality due to low treatment efficacy. Existing evidence indicates that inflammatory bowel disease (IBD) is associated with a higher risk of developing CRC. Many murine models of inflammation-related colon carcinogenesis (CAC) have been developed to study colon carcinogenesis and novel treatments. A commonly used model involves the combination of a single dose of azoxymethane (AOM), together with three cycles of the inflammatory agent dextran sodium sulfate (DSS) (5 days in drinking water followed by a two-week rest). Following this protocol, around 50% of the animals develop CRCs after 45 days and almost 100% of animals after 60 days. During CAC development, immune cells, cytokines, and other immune mediators are involved in both tumorigenesis and the elimination of cancer cells during immunotherapy. Thus, the study of mucosal immune responses (including lamina propria mononuclear cells and intraepithelial lymphocytes) is important to understand the role of the immune system during development and therapy in CRC. Single immune cell suspensions from lamina propria and epithelium can be purified combining selective tissue digestion and Percoll gradient centrifugation. Isolated cells can be characterized using flow cytometry by analyzing surface antigens or intracellular cytokines and cytotoxic mediators or employed for further investigations like comparative studies of mRNA expression, cell-proliferation assay, protein analysis, or even functional cytotoxicity assays. The CAC model is useful to study the involvement of immune cells not only during the carcinogenesis process but, in addition, during the treatment with novel immunotherapy protocols.

Purification of Leukemia-Derived Exosomes to Study Microenvironment Modulation.

Exosomes are membrane-enclosed vesicles released by different cell types into the extracellular space. As mediators of intercellular communication, they are involved in multiple physiological processes, but they are also associated with the pathogenesis of human malignancies including leukemia. Isolation of exosomes enables the characterization of their role in microenvironment modulation as well as their participation in disease pathology. A variety of strategies and techniques exists to purify exosomes from many biological fluids (e.g., blood, urine, and saliva). Here, we describe the efficient production of large quantities of exosomes from leukemic cell lines by using CELLine bioreactors based on two-compartment technology, as well as their isolation and purification by combining differential centrifugation and ultracentrifugation through a density gradient (17% OptiPrep™ cushion). Thus, exosomes are appropriately prepared for characterization by western blotting to detect exosome markers or imaging flow cytometry (ImageStream), and for downstream analyses such as the internalization in microenvironmental cells by confocal imaging or flow cytometry, methods which are also described in this chapter.

Detection of Antigen-Specific CD8+ T Cells in the Livers of HCV Core Transgenic Mice.

Hepatitis C virus-mediated immune suppression is an underlying feature leading to the establishment of viral persistence and chronic infection. In particular, HCV core protein has been shown to exhibit significant immunosuppressive activity of T cells and antigen presenting cells. Using an HCV core transgenic mouse system, in which liver hepatocytes express core protein, it is possible to study the effects of core-mediated immune suppression in vivo during viral infection. In this protocol, we describe the procedures for evaluating antigen-specific CD8+ T cell responses in response to recombinant adenovirus infection in HCV core transgenic mice.

Purification of Nongreen Plastids (Proplastids and Amyloplasts) from Angiosperms, and Isolation of Their Envelope Membranes.

Plastids, a wide family of plant specific organelles, exist in all plant cells in a number of different forms with different functions essential for plant life. Among them, chloroplasts are by far the more extensively studied owing to their central role in photosynthesis. However, other plastid family members, often referred to as nongreen plastids, play also major roles in the physiology of higher plants and could be better suited for studies of specific metabolic processes in heterotrophic plant cells. Unfortunately, serious technical problems are frequently encountered with separating intact, active nongreen plastids from contaminating membranes and mitochondria. Here, we provide detailed protocols suitable for the large scale preparation of intact and highly pure proplastids from cauliflower buds, as well as amyloplasts from sycamore cultured cells, and for the subsequent separation of their surrounding envelope membranes from the stroma and other plastid fractions. Both methods proved to be highly reliable and have been instrumental for in-depth investigations on biochemistry and physiology of nongreen plastids.

Isolation and Analysis of Mitochondrial Small RNAs from Rat Liver Tissue and HepG2 Cells.

The presence of noncoding RNAs, such as microRNAs (miRNAs), in mitochondria has been reported by several studies. The biological roles and functions of these mitochondrial miRNAs ("mitomiRs") have not been sufficiently characterized, but the mitochondrial localization of miRNAs has recently gained significance due to modified mitomiR-populations in certain states of diseases. Here, we describe the isolation and analysis of mitochondrial RNAs from rat liver tissue and HepG2 cells. The principle of the analysis is to prepare mitochondria by differential centrifugation. Cytosolic RNA contamination is eliminated by RNase A treatment followed by Percoll gradient purification and RNA extraction. Small RNA content is verified by capillary electrophoresis. Mitochondrial miRNAs are detected by qPCR following synthesis of cDNA. After qPCR-based mitomiR-profiling, the Normfinder algorithm is applied to identify the suitable reference miRNAs to use as normalizers for mitochondrial input and data analysis. The described procedure depicts a simple way of isolating and quantifying mitomiRs in tissue and cell culture samples.

Virus purification by CsCl density gradient using general centrifugation.

Virus purification by cesium chloride (CsCl) density gradient, which generally requires an expensive ultracentrifuge, is an essential technique in virology. Here, we optimized virus purification by CsCl density gradient using general centrifugation (40,000 × g, 2 h, 4 °C), which showed almost the same purification ability as conventional CsCl density gradient ultracentrifugation (100,000 × g, 1 h, 4 °C) using phages S13' and φEF24C. Moreover, adenovirus strain JM1/1 was also successfully purified by this method. We suggest that general centrifugation can become a less costly alternative to ultracentrifugation for virus purification by CsCl densiy gradient and will thus encourage research in virology.

Effect of rotor type on the separation of isotope-labeled and unlabeled Escherichia coli RNA by isopycnic density ultracentrifugation.

Separation of differentially isotope-labeled bacterial RNA by isopycnic density gradient centrifugation is a critical step in RNA-based stable isotope probing analyses, which help to link the structure and function of complex microbial communities. Using isotope-labeled Escherichia coli RNA, we showed that an 8 mL near-vertical rotor performed better than a 2 mL fixed-angle rotor, thereby corroborating current recommendations. Neither increased concentrations of formamide nor urea in the medium improved the separation results using the fixed-angle rotor.

Isolation of Nuclei and Nucleoli.

Here we describe methods for producing nuclei from Arabidopsis suspension cultures or root tips of Arabidopsis, wheat, or pea. These methods could be adapted for other species and cell types. The resulting nuclei can be further purified for use in biochemical or proteomic studies, or can be used for microscopy. We also describe how the nuclei can be used to obtain a preparation of nucleoli.

Isolation and Suborganellar Fractionation of Arabidopsis Chloroplasts.

Chloroplasts are structurally complex organelles containing ~2000-3000 proteins. They are delimited by a double membrane system or envelope, have an inner aqueous compartment called the stroma, and possess a second internal membrane system called the thylakoids. Thus, determining the suborganellar location of a chloroplast protein is vital to understanding or verifying its function. One way in which protein localization can be addressed is through fractionation. Here we present two rapid and simple methods that may be applied sequentially on the same day: (a) The isolation of intact chloroplasts from Arabidopsis thaliana plants that may be used directly (e.g., for functional studies such as protein import analysis), or for further processing as follows; (b) separation of isolated chloroplasts into three suborganellar fractions (envelope membranes, a soluble fraction containing stromal proteins, and the thylakoids). These methods are routinely used in our laboratory, and they provide a good yield of isolated chloroplasts and suborganellar fractions that can be used for various downstream applications.

Isolation of Chromoplasts and Suborganellar Compartments from Tomato and Bell Pepper Fruit.

Tomato is a model for fruit development and ripening. The isolation of intact plastids from this organism is therefore important for metabolic and proteomic analyses. Pepper, a species from the same family, is also of interest since it allows isolation of intact chromoplasts in large amounts. Here, we provide a detailed protocol for the isolation of tomato plastids at three fruit developmental stages, namely, nascent chromoplasts from the mature green stage, chromoplasts from an intermediate stage, and fully differentiated red chromoplasts. The method relies on sucrose density gradient centrifugations. It yields high purity organelles suitable for proteome analyses. Enzymatic and microscopy assays are summarized to assess purity and intactness. A method is also described for subfractionation of pepper chromoplast lipoprotein structures.

Leucoplast Isolation and Subfractionation.

Leucoplasts are colorless plastids of nonphotosynthetic plant tissues that support a variety of anabolic roles, particularly the biosynthesis of long-chain fatty acids in storage tissues of developing oil seeds. They also perform other important metabolic functions including the biosynthesis of amino acids and tetrapyrrole compounds. Leucoplasts use a complex set of membrane carriers and channels to actively translocate nuclear-encoded precursor proteins from the cytosol, while exchanging various metabolites with the cytosol. Leucoplast purification is a necessary prerequisite for detailed studies of their soluble (stromal) and membrane (envelope) (phospho)proteomes, as well as for achieving a detailed understanding of their metabolic capabilities, transport processes, and biogenesis. This chapter describes protocols for leucoplast purification from endosperm of developing castor oil seeds, and their subsequent subfractionation into envelope membrane and soluble stromal compartments for biochemical analysis.