Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice.
27 Related JoVE Articles!
Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates
Institutions: Cytori Therapeutics Inc, David Geffen School of Medicine at UCLA, David Geffen School of Medicine at UCLA, David Geffen School of Medicine at UCLA, David Geffen School of Medicine at UCLA.
In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.
Cellular Biology, Issue 79, Adipose Tissue, Stem Cells, Humans, Cell Biology, biology (general), enzymatic digestion, collagenase, cell isolation, Stromal Vascular Fraction (SVF), Adipose-derived Stem Cells, ASCs, lipoaspirate, liposuction
Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii
Institutions: The Geisel School of Medicine at Dartmouth.
Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii
has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii
by deleting the gene encoding the KU80 protein1,2
. The Δku80
strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro
and in vivo
and exhibit essentially a 100% frequency of homologous recombination. The Δku80
strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4
Here, we report methods for using type I and type II Δku80Δhxgprt
strains to advance gene targeting approaches in T. gondii
. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT
) selectable marker. The described gene targeting protocol can be used in a variety of ways in Δku80
strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii
and related significant human pathogens that cause malaria (Plasmodium
sp.) and cryptosporidiosis (Cryptosporidium
Infectious Diseases, Issue 77, Genetics, Microbiology, Infection, Medicine, Immunology, Molecular Biology, Cellular Biology, Biomedical Engineering, Bioengineering, Genomics, Parasitology, Pathology, Apicomplexa, Coccidia, Toxoplasma, Genetic Techniques, Gene Targeting, Eukaryota, Toxoplasma gondii, genetic manipulation, gene targeting, gene deletion, gene replacement, gene tagging, homologous recombination, DNA, sequencing
Enrichment and Purging of Human Embryonic Stem Cells by Detection of Cell Surface Antigens Using the Monoclonal Antibodies TG30 and GCTM-2
Human embryonic stem cells (hESC) can self-renew indefinitely in vitro
, and with the appropriate cues can be induced to differentiate into potentially all somatic cell lineages. Differentiated hESC derivatives can potentially be used in transplantation therapies to treat a variety of cell-degenerative diseases. However, hESC differentiation protocols usually yield a mixture of differentiated target and off-target cell types as well as residual undifferentiated cells. For the translation of differentiated hESC-derivatives from the laboratory to the clinic, it is important to be able to discriminate between undifferentiated (pluripotent) and differentiated cells, and generate methods to separate these populations. Safe application of hESC-derived somatic cell types can only be accomplished with pluripotent stem cell-free populations, as residual hESCs could induce tumors known as teratomas following transplantation. Towards this end, here we describe a methodology to detect pluripotency associated cell surface antigens with the monoclonal antibodies TG30 (CD9) and GCTM-2 via fluorescence activated cell sorting (FACS) for the identification of pluripotent TG30Hi
hESCs using positive selection. Using negative selection with our TG30/GCTM-2 FACS methodology, we were able to detect and purge undifferentiated hESCs in populations undergoing very early-stage differentiation (TG30Neg
). In a further study, pluripotent stem cell-free samples of differentiated TG30Neg
cells selected using our TG30/GCTM-2 FACS protocol did not form teratomas once transplanted into immune-compromised mice, supporting the robustness of our protocol. On the other hand, TG30/GCTM-2 FACS-mediated consecutive passaging of enriched pluripotent TG30Hi
hESCs did not affect their ability to self-renew in vitro
or their intrinsic pluripotency. Therefore, the characteristics of our TG30/GCTM-2 FACS methodology provide a sensitive assay to obtain highly enriched populations of hPSC as inputs for differentiation assays and to rid potentially tumorigenic (or residual) hESC from derivative cell populations.
Stem Cell Biology, Issue 82, Stem cells, cell surface antigens, antibodies, FACS, purging stem cells, differentiation, pluripotency, teratoma, human embryonic stem cells (hESC)
Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice
Institutions: San Raffaele Scientific Institute, Italian Cystic Fibrosis Research Foundation.
A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa
agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo
. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa
and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa
clinical strains in the agar-beads in vitro
, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa
RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa
colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.
Infection, Issue 85, Opportunistic Infections, Respiratory Tract Infections, Inflammation, Lung Diseases, Cystic Fibrosis, Pseudomonas aeruginosa
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
Application of Two-spotted Spider Mite Tetranychus urticae for Plant-pest Interaction Studies
Institutions: The University of Western Ontario, Instituto de Ciencias de la Vid y el Vino, Ghent University, University of Amsterdam.
The two-spotted spider mite, Tetranychus urticae
, is a ubiquitous polyphagous arthropod herbivore that feeds on a remarkably broad array of species, with more than 150 of economic value. It is a major pest of greenhouse crops, especially in Solanaceae
, tomatoes, eggplants, peppers, cucumbers, zucchini) and greenhouse ornamentals (e.g.
, roses, chrysanthemum, carnations), annual field crops (such as maize, cotton, soybean, and sugar beet), and in perennial cultures (alfalfa, strawberries, grapes, citruses, and plums)1,2
. In addition to the extreme polyphagy that makes it an important agricultural pest, T. urticae
has a tendency to develop resistance to a wide array of insecticides and acaricides that are used for its control3-7
is an excellent experimental organism, as it has a rapid life cycle (7 days at 27 °C) and can be easily maintained at high density in the laboratory. Methods to assay gene expression (including in situ
hybridization and antibody staining) and to inactivate expression of spider mite endogenous genes using RNA interference have been developed8-10
. Recently, the whole genome sequence of T. urticae
has been reported, creating an opportunity to develop this pest herbivore as a model organism with equivalent genomic resources that already exist in some of its host plants (Arabidopsis thaliana
and the tomato Solanum lycopersicum
. Together, these model organisms could provide insights into molecular bases of plant-pest interactions.
Here, an efficient method for quick and easy collection of a large number of adult female mites, their application on an experimental plant host, and the assessment of the plant damage due to spider mite feeding are described. The presented protocol enables fast and efficient collection of hundreds of individuals at any developmental stage (eggs, larvae, nymphs, adult males, and females) that can be used for subsequent experimental application.
Environmental Sciences, Issue 89, two-spotted spider mite, plant-herbivore interaction, Tetranychus urticae, Arabidopsis thaliana, plant damage analysis, herbivory, plant pests
Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae
Institutions: Rensselaer Polytechnic Institute.
has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae
has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.
Microbiology, Issue 92, Aging, mutations, genome instability, Saccharomyces cerevisiae, fluctuation test, magnetic sorting, mother cell, replicative aging
Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons
Institutions: University of Iowa Carver College of Medicine, University of Iowa Carver College of Medicine, EZ BioResearch LLC.
High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g.,
in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (ΔE-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.
Neuroscience, Issue 95, AP2, genotyping, glial feeder layer, mouse tail, neuronal culture, nucleic-acid extraction, PCR, tattoo, torsinA
Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples
Institutions: San Diego State University, DOE Joint Genome Institute, University of Colorado, University of Colorado.
The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.
Molecular Biology, Issue 94, virome, microbiome, metagenomics, metatranscriptomics, cystic fibrosis, mucosal-surface
Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology
Institutions: San Diego State University, University of California San Diego.
Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.
Environmental Sciences, Issue 93, dissolved organic carbon, particulate organic matter, nutrients, DAPI, SYBR, microbial metagenomics, viral metagenomics, marine environment
Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays
Institutions: University of Notre Dame, University of Notre Dame, University of Notre Dame, INRS-Institut Armand-Frappier, Indiana University, University of Notre Dame.
Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more “temperate swarmers” that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. “Wettability”, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux
operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.
Microbiology, Issue 98, Surface motility, Swarming, Imaging, Pseudomonas aeruginosa, Salmonella Typhimurium, Bacillus subtilis, Myxococcus xanthus, Flagella
Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity
Institutions: Northwestern University, Northwestern University, Northwestern University.
Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa
and dual-species biofilms of P. aeruginosa
and Escherichia coli
under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.
Bioengineering, Issue 97, microfluidic flow cell, chemical gradient, biofilm development, particle tracking, flow characterization, fluorescent tracer, solute transport
Naïve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures
Institutions: Technische Universität München, Technische Universität München.
Over the last decade, several adult stem cell populations have been identified in human skin 1-4
. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6
. These early studies described a multipotent precursor cell population from adult mammalian dermis 5
. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5
. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6
. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7
. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7
. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1
). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.
Stem Cell Biology, Issue 75, Cellular Biology, Molecular Biology, Anatomy, Physiology, Biomedical Engineering, Medicine, Dermatology, Cells, Cultured, Stem Cells, biology (general), Skin and Connective Tissue Diseases, Biological Phenomena, Adult stem cells, skin derived precursor cells, fibroblasts, sphere culture, skin-derived precursors, SKP, PCR, qPCR, immunocytochemistry, isolation, cell culture
The Use of Chemostats in Microbial Systems Biology
Institutions: New York University .
Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.
Environmental Sciences, Issue 80, Saccharomyces cerevisiae, Molecular Biology, Computational Biology, Systems Biology, Cell Biology, Genetics, Environmental Microbiology, Biochemistry, Chemostat, growth-rate, steady state, nutrient limitation, adaptive evolution
Trypsinizing and Subculturing Mammalian Cells
Institutions: Molecular Pathology Laboratory Network, Inc.
As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.
Basic Protocols, Issue 16, Current Protocols Wiley, Cell Culture, Cell Passaging, Trypsinizing Cells, Adherent Cells, Suspension Cells
Interview: Bioreactors and Surfaced-Modified 3D-Scaffolds for Stem Cell Research
Institutions: Karlsruhe Institute of Technology.
A Nature Editorial in 2003 asked the question "Good-bye, flat biology?" What does this question imply? In the past, many in vitro culture systems, mainly monolayer cultures, often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.
The last generation of CellChips -- micro-thermoformed containers -- a specific technology was developed, which offers the additional possibility to modify the whole surface of the 3D formed containers. This allows a surface-patterning on a submicron scale with distinct signalling molecules. Sensors and signal electrodes may be incorporated. Applications range from basic research in cell biology to toxicology and pharmacology. Using biodegradable polymers, clinical applications become a possibility. Furthermore, the last generation of micro-thermoformed chips has been optimized to allow for cheap mass production.
Cellular Biology, Issue 15, Interview, bioreactors, cell culture systems, 3D cell culture, stem cells
Reaggregate Thymus Cultures
Institutions: University of Birmingham .
Stromal cells within lymphoid tissues are organized into three-dimensional structures that provide a scaffold that is thought to control the migration and development of haemopoeitic cells. Importantly, the maintenance of this three-dimensional organization appears to be critical for normal stromal cell function, with two-dimensional monolayer cultures often being shown to be capable of supporting only individual fragments of lymphoid tissue function. In the thymus, complex networks of cortical and medullary epithelial cells act as a framework that controls the recruitment, proliferation, differentiation and survival of lymphoid progenitors as they undergo the multi-stage process of intrathymic T-cell development. Understanding the functional role of individual stromal compartments in the thymus is essential in determining how the thymus imposes self/non-self discrimination. Here we describe a technique in which we exploit the plasticity of fetal tissues to re-associate into intact three-dimensional structures in vitro
, following their enzymatic disaggregation. The dissociation of fetal thymus lobes into heterogeneous cellular mixtures, followed by their separation into individual cellular components, is then combined with the in vitro
re-association of these desired cell types into three-dimensional reaggregate structures at defined ratios, thereby providing an opportunity to investigate particular aspects of T-cell development under defined cellular conditions. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).
Immunology, Issue 18, Springer Protocols, Thymus, 2-dGuo, Thymus Organ Cultures, Immune Tolerance, Positive and Negative Selection, Lymphoid Development
Preparation of 2-dGuo-Treated Thymus Organ Cultures
Institutions: University of Birmingham .
In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo
can limit analysis of individual cellular components and particular stages of development. In vitro
culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro
. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro
with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).
Immunology, Issue 18, Springer Protocols, Thymus, 2-dGuo, Thymus Organ Cultures, Immune Tolerance, Positive and Negative Selection, Lymphoid Development
The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation
Institutions: Bio-Rad Laboratories, Inc.
It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.
Immunology, Issue 23, Primary Hematopoietic Cell Culture, Bone Marrow, Transfection, Electroporation, BioRad, IL-3
Culture and Maintenance of Human Embryonic Stem Cells
Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be fed every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.
Developmental Biology, Issue 34, Human embryonic stem cell, medium, hES, maintenance, thaw, ES cells, stem cell, cell culture, pluripotency, differentiation, passage, freeze
Strategies for Study of Neuroprotection from Cold-preconditioning
Institutions: The University of Chicago Medical Center.
Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by development of effective, easy to administer and safe preconditioning treatments. We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. Low-level pro-inflammatory mediator signaling changes over time are essential for cold-preconditioning neuroprotection. This signaling is consistent with the basic tenets of physiological conditioning hormesis, which require that irritative stimuli reach a threshold magnitude with sufficient time for adaptation to the stimuli for protection to become evident.
Accordingly, delineation of the immune signaling involved in cold-preconditioning neuroprotection requires that biological systems and experimental manipulations plus technical capacities are highly reproducible and sensitive. Our approach is to use hippocampal slice cultures as an in vitro
model that closely reflects their in vivo
counterparts with multi-synaptic neural networks influenced by mature and quiescent macroglia / microglia. This glial state is particularly important for microglia since they are the principal source of cytokines, which are operative in the femtomolar range. Also, slice cultures can be maintained in vitro
for several weeks, which is sufficient time to evoke activating stimuli and assess adaptive responses. Finally, environmental conditions can be accurately controlled using slice cultures so that cytokine signaling of cold-preconditioning can be measured, mimicked, and modulated to dissect the critical node aspects. Cytokine signaling system analyses require the use of sensitive and reproducible multiplexed techniques. We use quantitative PCR for TNF-α to screen for microglial activation followed by quantitative real-time qPCR array screening to assess tissue-wide cytokine changes. The latter is a most sensitive and reproducible means to measure multiple cytokine system signaling changes simultaneously. Significant changes are confirmed with targeted qPCR and then protein detection. We probe for tissue-based cytokine protein changes using multiplexed microsphere flow cytometric assays using Luminex technology. Cell-specific cytokine production is determined with double-label immunohistochemistry. Taken together, this brain tissue preparation and style of use, coupled to the suggested investigative strategies, may be an optimal approach for identifying potential targets for the development of novel therapeutics that could mimic the advantages of cold-preconditioning.
Neuroscience, Issue 43, innate immunity, hormesis, microglia, hippocampus, slice culture, immunohistochemistry, neural-immune, gene expression, real-time PCR
Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro
Institutions: The University of Chicago Medical Center, The University of Chicago Medical Center.
Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro
using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro
preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers.
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo
counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure
neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic
SD immune signaling.
Neuroscience, Issue 52, innate immunity, hormesis, microglia, T-cells, hippocampus, slice culture, gene expression, laser dissection microscopy, real-time qPCR, interferon-gamma
Aseptic Laboratory Techniques: Plating Methods
Institutions: University of California, Los Angeles .
Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories
(BMBL) as well as Material Safety Data Sheets
(MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection
(ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to:
● Perform plating procedures without contaminating media.
● Isolate single bacterial colonies by the streak-plating method.
● Use pour-plating and spread-plating methods to determine the concentration of bacteria.
● Perform soft agar overlays when working with phage.
● Transfer bacterial cells from one plate to another using the replica-plating procedure.
● Given an experimental task, select the appropriate plating method.
Basic Protocols, Issue 63, Streak plates, pour plates, soft agar overlays, spread plates, replica plates, bacteria, colonies, phage, plaques, dilutions
Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer
Institutions: Life Technologies , Life Technologies .
Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide different information. Flow cytometry gives quantitative multi-parametric information about physical characteristics and staining or expression, but doesn't allow for visualization. Stand-alone fluorescence microscopy provides visual data, but doesn't allow for straightforward quantitative measurements1
Image-based cytometry bridges the gap between these two methods, enabling the quick visualization and simultaneous quantitative analysis of thousands of cells in heterogeneous populations2
. Here, we present a method for performing cell viability and green fluorescent protein (GFP) expression assays using the Tali Image-Based Cytometer3
. The Tali instrument is a 3-channel (bright field, green fluorescence, red fluorescence) benchtop assay platform that offers several advantages over flow cytometry and fluorescence microscopy. The Tali cytometer is less expensive, takes up less bench space, requires less maintenance, and the work flow has been simplified so that the operation and analysis is much simpler and quicker. The Tali cytometer is capable of performing a range of suspension cell-based assays, including GFP and red fluorescent protein (RFP) expression, apoptosis4-6
and cell viability analysis with propidium iodide (PI)7-11
Here, we demonstrate the use of the Tali instrument in performing a cell viability assay in cells expressing GFP. GFP-transduced cells are stained using the Tali Viability Kit - Dead Cell Red. The cells are then pipetted into a Tali Cellular Analysis Slide and loaded into the cytometer. Bright field, red fluorescence and green fluorescence images are captured and analyzed using assay specific algorithms. Histograms are then generated to display cell size, PI fluorescence intensity, and GFP fluorescence intensity. These parameters can then be thresholded to home in on a specific cell population.
A side-by side comparison of the Tali Image-Based Cytometer and traditional flow cytometry demonstrates that the two methods provide comparable data regarding cell viability and protein expression. However, the Tali instrument provides additional visual information about the cell population that cannot be obtained using a flow cytometer.
Cell Biology, Issue 57, cytometry, imaging, image-based, cell viability, apoptosis, cell counting, expression assay, Dead-Cell Red, propidium iodide, PI, GFP expression, RFP expression, cell analysis, fluorescence protein expression
Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung
Institutions: University of Liverpool , University of Liverpool .
There is growing concern about the relevance of in vitro
antimicrobial susceptibility tests when applied to isolates of P. aeruginosa
from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1
. However, during chronic lung infections in CF, P. aeruginosa
populations exist in biofilms and there is evidence that the environment is largely microaerophilic2
. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3
Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa
growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6
. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa
based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
An ASM assay was developed in a microtitre plate format. P. aeruginosa
biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa
isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a >128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3
. Several in vitro
models have been used previously to study P. aeruginosa
. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9
. In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2
and affect antibiotic susceptibility10
. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.
Immunology, Issue 64, Microbiology, Pseudomonas aeruginosa, antimicrobial susceptibility, artificial sputum media, lung infection, cystic fibrosis, diagnostics, plankton
Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes
Institutions: Roswell Park Cancer Institute, University of Pennsylvania , SciGro, Inc., University of Pennsylvania .
Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro
and in vivo
Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
stem and progenitor cell quiescence, proliferation and/or differentiation6-8
antigen-driven membrane transfer9
and/or precursor cell proliferation3,4,10-18
immune regulatory and effector cell function1,18-21
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11
. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18
. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
Failure to achieve bright, uniform, reproducible labeling
. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.
Suboptimal fluorochrome combinations and/or failure to include critical compensation controls
. Tracking dye fluorescence is typically 102
times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.
Failure to obtain a good fit with peak modeling software
. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.
Cellular Biology, Issue 70, Molecular Biology, Cell tracking, PKH26, CFSE, membrane dyes, dye dilution, proliferation modeling, lymphocytes
Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins
Institutions: San Diego State University, San Diego State University, San Diego State University, San Diego State University, San Diego State University, Argonne National Laboratory, Broad Institute.
Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.
Immunology, Issue 100, phenomics, phage, viral metagenome, Multi-phenotype Assay Plates (MAPs), continuous culture, metabolomics