Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as “the scraping method” and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed “the trypsin method.” This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).
22 Related JoVE Articles!
Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression
Institutions: University of Southern California, University College London.
Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2
. Whilst stage I tumors have 5-year survival rates >85%, survival rates for stage III/IV disease are <40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5
. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g.
age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF) cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT
) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC
oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.
Cancer Biology, Issue 66, Medicine, Tissue Engineering, three-dimensional cultures, stromal-epithelial interactions, epithelial ovarian cancer, ovarian surface epithelium, ovarian fibroblasts, tumor initiation
The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins
Institutions: Children's Hospital of Pittsburgh of UPMC, University of Pittsburgh School of Medicine.
Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e.
endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.
Basic Protocol, Issue 82, Endocytosis, recycling, plasma membrane, cell surface, EZLink, Sulfo-NHS-SS-Biotin, L-Glutathione, GSH, thiol group, disulfide bond, epithelial cells, cell polarization
Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium
Institutions: Washington University School of Medicine, Washington University School of Medicine.
The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. During infection of the urinary bladder, polymorphonuclear leukocytes (PMN; neutrophils) migrate from the bloodstream and traverse the bladder epithelium. Failure to resolve infection in the absence of a neutrophilic response demonstrates the importance of PMN in bladder defense. To facilitate colonization of the bladder epithelium, uropathogenic Escherichia coli
(UPEC), the causative agent of the majority of urinary tract infections (UTIs), dampen the acute inflammatory response using a variety of partially defined mechanisms. To further investigate the interplay between host and bacterial pathogen, we developed an in vitro
model of this aspect of the innate immune response to UPEC. In the transuroepithelial neutrophil migration assay, a variation on the Boyden chamber, cultured bladder epithelial cells are grown to confluence on the underside of a permeable support. PMN are isolated from human venous blood and are applied to the basolateral side of the bladder epithelial cell layers. PMN migration representing the physiologically relevant basolateral-to-apical direction in response to bacterial infection or chemoattractant molecules is enumerated using a hemocytometer. This model can be used to investigate interactions between UPEC and eukaryotic cells as well as to interrogate the molecular requirements for the traversal of bladder epithelia by PMN. The transuroepithelial neutrophil migration model will further our understanding of the initial inflammatory response to UPEC in the bladder.
Immunology, Issue 81, uropathogenic Escherichia coli, neutrophil, bladder epithelium, neutrophil migration, innate immunity, urinary tract infection
Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay
Institutions: Murdoch Childrens Research Institute, Murdoch Childrens Research Institute, The University of Melbourne, The University of Melbourne.
Adherence of Streptococcus pneumoniae
(the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro
adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal colonization. The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius
on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.
Immunology, Issue 86, Gram-Positive Bacterial Infections, Pneumonia, Bacterial, Lung Diseases, Respiratory Tract Infections, Streptococcus pneumoniae, adherence, colonization, probiotics, Streptococcus salivarius, In Vitro assays
Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model
Institutions: United States Department of Agriculture, University of Maryland.
The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic alterations originated during preimplantation development on subsequent fetal development and adult health. The use of an effective and consistent embryo transfer technique is crucial to enhance the generation of genetically modified animals and to determine the effect of different treatments on implantation rates and survival to term. Embryos at the blastocyst stage are usually transferred by uterine transfer, performing a puncture in the uterine wall to introduce the embryo manipulation pipette. The orifice performed in the uterus does not close after the pipette has been withdrawn, and the embryos can outflow to the abdominal cavity due to the positive pressure of the uterus. The puncture can also produce a hemorrhage that impairs implantation, blocks the transfer pipette and may affect embryo development, especially when embryos without zona
are transferred. Consequently, this technique often results in very variable and overall low embryo survival rates. Avoiding these negative effects, utero-tubal embryo transfer take advantage of the utero-tubal junction as a natural barrier that impedes embryo outflow and avoid the puncture of the uterine wall. Vasectomized males are required for obtaining pseudopregnant recipients. A technique to perform vasectomy is described as a complement to the utero-tubal embryo transfer.
Basic Protocols, Issue 84, blastocyst, chimera, lentivirus, uterine transfer, oviductal transfer, utero-tubal transfer
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),
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro
. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro
replication of HIV-1 as influenced by the gag
gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag
gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro
replication of chronically derived gag-pro
sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice
Institutions: Case Western Reserve University.
Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of infection-induced immunopathology. Although Th17 cells are implicated in antifungal defense, their role in immunopathology is unclear. This study presents a method for establishing oral Th17 immunopathology associated with oral candidal infection in immunodeficient mice. The method is based on reconstituting lymphopenic mice with in vitro
cultured Th17 cells, followed by oral infection with Candida albicans
Results show that unrestrained Th17 cells result in inflammation and pathology, and is associated with several measurable read-outs including weight loss, pro-inflammatory cytokine production, tongue histopathology and mortality, showing that this model may be valuable in studying OPC immunopathology. Adoptive transfer of regulatory cells (Tregs
) controls and reduces the inflammatory response, showing that this model can be used to test new strategies to counteract oral inflammation. This model may also be applicable in studying oral Th17 immunopathology in general in the context of other oral diseases.
Medicine, Issue 96, Th17, Treg, mouse model, oral inflammation, Candida, oral infection and immunopathology
Isolation of Leukocytes from the Human Maternal-fetal Interface
Institutions: NICHD/NIH/DHHS, University of Michigan, Michigan State University, Wayne State University, Wayne State University School of Medicine, Wayne State University School of Medicine.
Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro
leukocyte functionality (i.e.
, phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.
), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.
Immunology, Issue 99, Accutase, Decidua Basalis, Decidua Parietalis, Flow Cytometry, Immunophenotyping, Pregnancy
In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration
Institutions: Harvard Medical School, MGH for Children, Massachusetts General Hospital.
Mucosal surfaces serve as protective barriers against pathogenic organisms. Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils. This process has the potential to be destructive to tissues if excessive or held in an unresolved state. Cocultured in vitro
models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration. This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil. Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface. The in vitro
model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa
(PAO1). Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli
(MC1000). The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls. This in vitro
model system can be manipulated and applied to other mucosal surfaces. Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections. A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro
coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.
Infection, Issue 83, Cellular Biology, Epithelium, Neutrophils, Pseudomonas aeruginosa, Respiratory Tract Diseases, Neutrophils, epithelial barriers, pathogens, transmigration
Mouse Embryonic Development in a Serum-free Whole Embryo Culture System
Institutions: University of Georgia, University of Georgia.
Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2
/ 5% CO2
in a rolling bottle culture apparatus at 37 °C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero
. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.
Developmental Biology, Issue 85, mouse embryo, mid-gestation, serum-free, defined media, roller culture, organogenesis, development
An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection
Institutions: Radboud university medical center.
Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology. Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro
model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.
Immunology, Issue 82, Blood Cells, Respiratory Syncytial Virus, Human, Respiratory Tract Infections, Paramyxoviridae Infections, Models, Immunological, Immunity, HRSV culture, purification, quantification, PBMC isolation, stimulation, inflammatory pathways
Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation
Institutions: F. Edward Hebert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD.
A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1
. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro
and in vivo 2
. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.
Cellular Biology, Issue 32, Mammospheres, HC11, lactogenic differentiation, mammary
Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching
Institutions: Childrens Hospital Los Angeles.
Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.
Developmental Biology, Issue 40, lung, mice, culture
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
Invasion of Human Cells by a Bacterial Pathogen
Institutions: University of Bath.
Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus
. The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus
would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2
at 37°C. For S. aureus
this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 μl tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.
Infection, Issue 49, Bacterial pathogen, host cell invasion, Staphylococcus aureus, invasin
In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells
Institutions: Groupe de Recherche sur les Maladies Infectieuses du Porc GREMIP, Faculte de medecine veterinaire.
To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1
. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2
. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4
. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli
, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA
(F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli
, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6
Infection, Issue 51, adhesion, invasion, bacteria, cell culture, microbiology, cell biology
Mouse Model of Surgically-induced Endometriosis by Auto-transplantation of Uterine Tissue
Institutions: University of Missouri, University of Missouri.
Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of lesions, and/or chronic pharmaceutical management of pain and infertility symptoms. The cost associated with endometriosis has been estimated at 22 billion dollars per year in the United States1
. To further our understanding of mechanisms underlying this enigmatic disease, animal models have been employed. Primates spontaneously develop endometriosis and therefore primate models most closely resemble the disease in women. Rodent models, however, are more cost effective and readily available2
. The model that we describe here involves an autologous transfer of uterine tissue to the intestinal mesentery (Figure 1) and was first developed in the rat3
and later transferred to the mouse4
. The goal of the autologous rodent model of surgically-induced endometriosis is to mimic the disease in women. We and others have previously shown that the altered gene expression pattern observed in endometriotic lesions from mice or rats mirrors that observed in women with the disease5,6
. One advantage of performing the surgery in the mouse is that the abundance of transgenic mouse strains available can aid researchers in determining the role of specific components important in the establishment and growth of endometriosis. An alternative model in which excised human endometrial fragments are introduced to the peritoneum of immunocompromised mice is also widely used but is limited by the lack of a normal immune system which is thought to be important in endometriosis2,7
. Importantly, the mouse model of surgically induced endometriosis is a versatile model that has been used to study how the immune system8
and environmental factors11,12
affect endometriosis as well as the effects of endometriosis on fertility13
Medicine, Issue 59, mouse, rat, endometriosis, surgery, uterus, ectopic, endometriotic lesion
A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants
Institutions: European Institute of Oncology, European Institute of Oncology, Ospedale Policlinico di Milano.
Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper homeostasis directly depends on these interactions, as they shape an entire immunological response inducing tolerance against food antigens while at the same time mounting effective immune responses against pathogenic microbes accidentally ingested with food.
Intestinal homeostasis is preserved also through various complex interactions between the microbiota (including food-associated beneficial bacterial strains) and the host, that regulate the attachment/degradation of mucus, the production of antimicrobial peptides by the epithelial barrier, and the "education" of epithelial cells' that controls the tolerogenic or immunogenic phenotype of unique, gut-resident lymphoid cells' populations. These interactions have been so far very difficult to reproduce with in vitro
assays using either cultured cell lines or peripheral blood mononuclear cells. In addition, mouse models differ substantially in components of the intestinal mucosa (mucus layer organization, commensal bacteria community) with respect to the human gut. Thus, studies of a variety of treatments to be brought in the clinics for important stress-related or pathological conditions such as irritable bowel syndrome, inflammatory bowel disease or colorectal cancer have been difficult to carry out.
To address these issues, we developed a novel system that enables us to stimulate explants of human intestinal mucosa that retain their in situ
conditioning by the host microbiota and immune response, in a polarized fashion. Polarized apical stimulation is of great importance for the outcome of the elicited immune response. It has been repeatedly shown that the same stimuli can produce completely different responses when they bypass the apical face of the intestinal epithelium, stimulating epithelial cells basolaterally or coming into direct contact with lamina propria components, switching the phenotype from tolerogenic to immunogenic and causing unnecessary and excessive inflammation in the area.
We achieved polarized stimulation by gluing a cave cylinder which delimited the area of stimulation on the apical face of the mucosa as will be described in the protocol. We used this model to examine, among others, differential effects of three different Lactobacilli
strains. We show that this model system is very powerful to assess the immunomodulatory properties of probiotics in healthy and disease conditions.
Microbiology, Issue 75, Cellular Biology, Medicine, Molecular Biology, Biomedical Engineering, Anatomy, Physiology, Bacteria, Tissue Engineering, Tissue culture, intestinal mucosa, polarized stimulation, probiotics, explants, Lactobacilli, microbiota, cell culture
Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells
Institutions: KU Leuven.
Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+
-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3
that initiate the propagation of the Ca2+
-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+
-wave propagation are provided by gap junction channels through the direct transfer of IP3
and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+
-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+
-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 μm. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+
-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.
Cellular Biology, Issue 77, Molecular Biology, Medicine, Biomedical Engineering, Biophysics, Immunology, Ophthalmology, Gap Junctions, Connexins, Connexin 43, Calcium Signaling, Ca2+, Cell Communication, Paracrine Communication, Intercellular communication, calcium wave propagation, gap junctions, hemichannels, endothelial cells, cell signaling, cell, isolation, cell culture
Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages
Institutions: Kingsborough Community College, University of Texas at Austin, Kean University.
The positive role of fatty acids in the prevention and alleviation of non-human and human diseases have been and continue to be extensively documented. These roles include influences on infectious and non-infectious diseases including prevention of inflammation as well as mucosal immunity to infectious diseases. Cholera is an acute intestinal illness caused by the bacterium Vibrio cholerae
. It occurs in developing nations and if left untreated, can result in death. While vaccines for cholera exist, they are not always effective and other preventative methods are needed. We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin using mouse BALB/C macrophages and human intestinal epithelial cells, respectively. We solubilized the above fatty acids and used cell proliferation assays to determine the concentration ranges and specific concentrations of the fatty acids that are not detrimental to human intestinal epithelial cell viability. We solubilized cholera toxin and used it in an assay to determine the concentration ranges and specific concentrations of cholera toxin that do not statistically decrease cell viability in BALB/C macrophages.
We found the optimum fatty acid concentrations to be between 1-5 ng/μl, and that for cholera toxin to be < 30 ng per treatment. This data may aid future studies that aim to find a protective mucosal role for fatty acids in prevention or alleviation of cholera infections.
Infection, Issue 75, Medicine, Immunology, Infectious Diseases, Microbiology, Molecular Biology, Cellular Biology, Biochemistry, Bioengineering, Bacterial Infections and Mycoses, Mucosal immunity, oleic acid, linoleic acid, linolenic acid, cholera toxin, cholera, fatty acids, tissue culture, MTT assay, mouse, animal model
Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface
Institutions: Wayne State University School of Medicine, The University of Adelaide, Wayne State University School of Medicine, NICHD/NIH/DHHS.
Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (>70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro
functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.
Immunology, Issue 99, Decidua, Dissociation, Isolation, Leukocytes, Myometrium, Placenta, Pregnancy, Uterus