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Pubmed Article
Multivalent presentation of MPL by porous silicon microparticles favors T helper 1 polarization enhancing the anti-tumor efficacy of doxorubicin nanoliposomes.
PLoS ONE
PUBLISHED: 01-01-2014
Porous silicon (pSi) microparticles, in diverse sizes and shapes, can be functionalized to present pathogen-associated molecular patterns that activate dendritic cells. Intraperitoneal injection of MPL-adsorbed pSi microparticles, in contrast to free MPL, resulted in the induction of local inflammation, reflected in the recruitment of neutrophils, eosinophils and proinflammatory monocytes, and the depletion of resident macrophages and mast cells at the injection site. Injection of microparticle-bound MPL resulted in enhanced secretion of the T helper 1 associated cytokines IFN-? and TNF-? by peritoneal exudate and lymph node cells in response to secondary stimuli while decreasing the anti-inflammatory cytokine IL-10. MPL-pSi microparticles independently exhibited anti-tumor effects and enhanced tumor suppression by low dose doxorubicin nanoliposomes. Intravascular injection of the MPL-bound microparticles increased serum IL-1? levels, which was blocked by the IL-1 receptor antagonist Anakinra. The microparticles also potentiated tumor infiltration by dendritic cells, cytotoxic T lymphocytes, and F4/80+ macrophages, however, a specific reduction was observed in CD204+ macrophages.
Authors: Shelley B. Weisser, Nico van Rooijen, Laura M. Sly.
Published: 08-01-2012
ABSTRACT
Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFNγ or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).
27 Related JoVE Articles!
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Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy
Authors: Rachel Lubong Sabado, Elizabeth Miller, Meredith Spadaccia, Isabelita Vengco, Farah Hasan, Nina Bhardwaj.
Institutions: NYU Langone Medical Center, NYU Langone Medical Center.
While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be established. The method described below, prepared in accordance with Good Manufacturing Process (GMP) guidelines, is an optimization of the most common ex vivo preparation method for generating large numbers of DCs for clinical studies 2. Our method utilizes the synthetic TLR 3 agonist Polyinosinic-Polycytidylic Acid-poly-L-lysine Carboxymethylcellulose (Poly-ICLC) to stimulate the DCs. Our previous study established that Poly-ICLC is the most potent individual maturation stimulus for human DCs as assessed by an upregulation of CD83 and CD86, induction of interleukin-12 (IL-12), tumor necrosis factor (TNF), interferon gamma-induced protein 10 (IP-10), interleukmin 1 (IL-1), and type I interferons (IFN), and minimal interleukin 10 (IL-10) production. DCs are differentiated from frozen peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis. PBMCs are isolated by Ficoll gradient centrifugation and frozen in aliquots. On Day 1, PBMCs are thawed and plated onto tissue culture flasks to select for monocytes which adhere to the plastic surface after 1-2 hr incubation at 37 °C in the tissue culture incubator. After incubation, the lymphocytes are washed off and the adherent monocytes are cultured for 5 days in the presence of interleukin-4 (IL-4) and granulocyte macrophage-colony stimulating factor (GM-CSF) to differentiate to immature DCs. On Day 6, immature DCs are pulsed with the keyhole limpet hemocyanin (KLH) protein which serves as a control for the quality of the vaccine and may boost the immunogenicity of the vaccine 3. The DCs are stimulated to mature, loaded with peptide antigens, and incubated overnight. On Day 7, the cells are washed, and frozen in 1 ml aliquots containing 4 - 20 x 106 cells using a controlled-rate freezer. Lot release testing for the batches of DCs is performed and must meet minimum specifications before they are injected into patients.
Cancer Biology, Issue 78, Medicine, Immunology, Molecular Biology, Cellular Biology, Biomedical Engineering, Anatomy, Physiology, Dendritic Cells, Immunotherapy, dendritic cell, immunotherapy, vaccine, cell, isolation, flow cytometry, cell culture, clinical techniques
50085
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Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation
Authors: Karen L. Joyce, Will Morgan, Robert Greenberg, Meera G. Nair.
Institutions: University of Pennsylvania , University of Pennsylvania .
Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.
Immunology, Issue 64, Infection, Microbiology, helminth, parasite, mouse, Th2, lung, inflammation, granuloma, alternative activation, macrophage
3905
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In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis
Authors: Guillaume Bierry, Sophie Lefevre, Jean-Louis Dietemann, François Jehl.
Institutions: University Hospital of Strasbourg, University of Strasbourg, University Hospital of Strasbourg.
Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are intensively and increasingly recruited in septic joints from the early phases of infection and the infiltration is supposed to regress once efficient removal of the pathogens is obtained. The ability to identify in vivo macrophage activity in an infected joint can therefore provide two main applications: early detection of acute synovitis and monitoring of therapy. In vivo noninvasive detection of macrophages can be performed with magnetic resonance imaging using iron nanoparticles such as ultrasmall superparamagnetic iron oxide (USPIO). After intravascular or intraarticular administration, USPIO are specifically phagocytized by activated macrophages, and, due to their magnetic properties, induce signal changes in tissues presenting macrophage infiltration. A quantitative evaluation of the infiltrate is feasible, as the area with signal loss (number of dark pixels) observed on gradient echo MR images after particles injection is correlated with the amount of iron within the tissue and therefore reflects the number of USPIO-loaded cells. We present here a protocol to perform macrophage imaging using USPIO-enhanced MR imaging in an animal model of septic arthritis, allowing an initial and longitudinal in vivo noninvasive evaluation of macrophages infiltration and an assessment of therapy action.
Medicine, Issue 80, Biomedical Engineering, Anatomy, Physiology, Molecular Biology, Diagnostic Imaging, Musculoskeletal System, Bacterial Infections and Mycoses, Macrophage, MR imaging, infection, arthritis, USPIO, imaging, clinical techniques
50296
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Micropunching Lithography for Generating Micro- and Submicron-patterns on Polymer Substrates
Authors: Anirban Chakraborty, Xinchuan Liu, Cheng Luo.
Institutions: University of Texas at Arlington .
Conducting polymers have attracted great attention since the discovery of high conductivity in doped polyacetylene in 19771. They offer the advantages of low weight, easy tailoring of properties and a wide spectrum of applications2,3. Due to sensitivity of conducting polymers to environmental conditions (e.g., air, oxygen, moisture, high temperature and chemical solutions), lithographic techniques present significant technical challenges when working with these materials4. For example, current photolithographic methods, such as ultra-violet (UV), are unsuitable for patterning the conducting polymers due to the involvement of wet and/or dry etching processes in these methods. In addition, current micro/nanosystems mainly have a planar form5,6. One layer of structures is built on the top surfaces of another layer of fabricated features. Multiple layers of these structures are stacked together to form numerous devices on a common substrate. The sidewall surfaces of the microstructures have not been used in constructing devices. On the other hand, sidewall patterns could be used, for example, to build 3-D circuits, modify fluidic channels and direct horizontal growth of nanowires and nanotubes. A macropunching method has been applied in the manufacturing industry to create macropatterns in a sheet metal for over a hundred years. Motivated by this approach, we have developed a micropunching lithography method (MPL) to overcome the obstacles of patterning conducting polymers and generating sidewall patterns. Like the macropunching method, the MPL also includes two operations (Fig. 1): (i) cutting; and (ii) drawing. The "cutting" operation was applied to pattern three conducting polymers4, polypyrrole (PPy), Poly(3,4-ethylenedioxythiophen)-poly(4-styrenesulphonate) (PEDOT) and polyaniline (PANI). It was also employed to create Al microstructures7. The fabricated microstructures of conducting polymers have been used as humidity8, chemical8, and glucose sensors9. Combined microstructures of Al and conducting polymers have been employed to fabricate capacitors and various heterojunctions9,10,11. The "cutting" operation was also applied to generate submicron-patterns, such as 100- and 500-nm-wide PPy lines as well as 100-nm-wide Au wires. The "drawing" operation was employed for two applications: (i) produce Au sidewall patterns on high density polyethylene (HDPE) channels which could be used for building 3D microsystems12,13,14, and (ii) fabricate polydimethylsiloxane (PDMS) micropillars on HDPE substrates to increase the contact angle of the channel15.
Mechanical Engineering, Issue 65, Physics, micropunching lithography, conducting polymers, nanowires, sidewall patterns, microlines
3725
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Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer
Authors: Elizabeth S. Nakasone, Hanne A. Askautrud, Mikala Egeblad.
Institutions: Watson School of Biological Sciences, Cold Spring Harbor Laboratory, University of Oslo and Oslo University Hospital.
The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.
Cancer Biology, Issue 73, Medicine, Molecular Biology, Cellular Biology, Biomedical Engineering, Genetics, Oncology, Pharmacology, Surgery, Tumor Microenvironment, Intravital imaging, chemotherapy, Breast cancer, time-lapse, mouse models, cancer cell death, stromal cell migration, cancer, imaging, transgenic, animal model
50088
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Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors
Authors: Jae Youn Hwang, Daniel L. Farkas, Lali K. Medina-Kauwe.
Institutions: University of Southern California, Cedars-Sinai Medical Center, University of California, Los Angeles.
The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. HerDox is formed by non-covalent self-assembly of a tumor targeted cell penetration protein with the chemotherapy agent, doxorubicin, via a small nucleic acid linker. A combination of electrophilic, intercalation, and oligomerization interactions facilitate self-assembly into round 10-20 nm particles. HerDox exhibits stability in blood as well as in extended storage at different temperatures. Systemic delivery of HerDox in tumor-bearing mice results in tumor-cell death with no detectable adverse effects to non-tumor tissue, including the heart and liver (which undergo marked damage by untargeted doxorubicin). HER2 elevation facilitates targeting to cells expressing the human epidermal growth factor receptor, hence tumors displaying elevated HER2 levels exhibit greater accumulation of HerDox compared to cells expressing lower levels, both in vitro and in vivo. Fluorescence intensity imaging combined with in situ confocal and spectral analysis has allowed us to verify in vivo tumor targeting and tumor cell penetration of HerDox after systemic delivery. Here we detail our methods for assessing tumor targeting via multimode imaging after systemic delivery.
Biomedical Engineering, Issue 76, Cancer Biology, Medicine, Bioengineering, Molecular Biology, Cellular Biology, Biochemistry, Nanotechnology, Nanomedicine, Drug Delivery Systems, Molecular Imaging, optical imaging devices (design and techniques), HerDox, Nanoparticle, Tumor, Targeting, Self-Assembly, Doxorubicin, Human Epidermal Growth Factor, HER, HER2+, Receptor, mice, animal model, tumors, imaging
50396
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Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?
Authors: Julia Y. Ljubimova, Hui Ding, Jose Portilla-Arias, Rameshwar Patil, Pallavi R. Gangalum, Alexandra Chesnokova, Satoshi Inoue, Arthur Rekechenetskiy, Tala Nassoura, Keith L. Black, Eggehard Holler.
Institutions: Cedars-Sinai Medical Center.
Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this diversity, personalized medicine is developed for silencing malignancy associated genes. Nano drugs fit these needs by targeting tumor and delivering antisense oligonucleotides for silencing of genes. As drugs for the treatment are often administered repeatedly, absence of toxicity and negligible immune response are desirable. In the example presented here, a nano medicine is synthesized from the biodegradable, non-toxic and non-immunogenic platform polymalic acid by controlled chemical ligation of antisense oligonucleotides and tumor targeting molecules. The synthesis and treatment is exemplified for human Her2-positive breast cancer using an experimental mouse model. The case can be translated towards synthesis and treatment of other tumors.
Chemistry, Issue 88, Cancer treatment, personalized medicine, polymalic acid, nanodrug, biopolymer, targeting, host compatibility, biodegradability
50668
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Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma
Authors: Ayman I. Omar.
Institutions: Southern Illinois University School of Medicine.
A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1. Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9. Bevacizumab however failed to prolong overall survival in a recent phase III trial26. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians’ choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10. There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.
Medicine, Issue 92, Tumor Treating Fields, TTF System, TTF Therapy, Recurrent Glioblastoma, Bevacizumab, Brain Tumor
51638
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Fabrication And Characterization Of Photonic Crystal Slow Light Waveguides And Cavities
Authors: Christopher Paul Reardon, Isabella H. Rey, Karl Welna, Liam O'Faolain, Thomas F. Krauss.
Institutions: University of St Andrews.
Slow light has been one of the hot topics in the photonics community in the past decade, generating great interest both from a fundamental point of view and for its considerable potential for practical applications. Slow light photonic crystal waveguides, in particular, have played a major part and have been successfully employed for delaying optical signals1-4 and the enhancement of both linear5-7 and nonlinear devices.8-11 Photonic crystal cavities achieve similar effects to that of slow light waveguides, but over a reduced band-width. These cavities offer high Q-factor/volume ratio, for the realization of optically12 and electrically13 pumped ultra-low threshold lasers and the enhancement of nonlinear effects.14-16 Furthermore, passive filters17 and modulators18-19 have been demonstrated, exhibiting ultra-narrow line-width, high free-spectral range and record values of low energy consumption. To attain these exciting results, a robust repeatable fabrication protocol must be developed. In this paper we take an in-depth look at our fabrication protocol which employs electron-beam lithography for the definition of photonic crystal patterns and uses wet and dry etching techniques. Our optimised fabrication recipe results in photonic crystals that do not suffer from vertical asymmetry and exhibit very good edge-wall roughness. We discuss the results of varying the etching parameters and the detrimental effects that they can have on a device, leading to a diagnostic route that can be taken to identify and eliminate similar issues. The key to evaluating slow light waveguides is the passive characterization of transmission and group index spectra. Various methods have been reported, most notably resolving the Fabry-Perot fringes of the transmission spectrum20-21 and interferometric techniques.22-25 Here, we describe a direct, broadband measurement technique combining spectral interferometry with Fourier transform analysis.26 Our method stands out for its simplicity and power, as we can characterise a bare photonic crystal with access waveguides, without need for on-chip interference components, and the setup only consists of a Mach-Zehnder interferometer, with no need for moving parts and delay scans. When characterising photonic crystal cavities, techniques involving internal sources21 or external waveguides directly coupled to the cavity27 impact on the performance of the cavity itself, thereby distorting the measurement. Here, we describe a novel and non-intrusive technique that makes use of a cross-polarised probe beam and is known as resonant scattering (RS), where the probe is coupled out-of plane into the cavity through an objective. The technique was first demonstrated by McCutcheon et al.28 and further developed by Galli et al.29
Physics, Issue 69, Optics and Photonics, Astronomy, light scattering, light transmission, optical waveguides, photonics, photonic crystals, Slow-light, Cavities, Waveguides, Silicon, SOI, Fabrication, Characterization
50216
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Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors
Authors: Naama Massad-Ivanir, Giorgi Shtenberg, Ester Segal.
Institutions: Technion - Israel Institute of Technology, Technion - Israel Institute of Technology, Technion - Israel Institute of Technology.
A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g. by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-Pérot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a 'real time' observation of bacteria attachment. The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.
Bioengineering, Issue 81, analytical chemistry, silicon materials, microbiology, optical materials, Porous Si, optical biosensor, bacteria detection, label-free biosensor, nanostructure, E. coli bacteria
50805
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Single Plane Illumination Module and Micro-capillary Approach for a Wide-field Microscope
Authors: Thomas Bruns, Sarah Schickinger, Herbert Schneckenburger.
Institutions: Aalen University.
A module for light sheet or single plane illumination microscopy (SPIM) is described which is easily adapted to an inverted wide-field microscope and optimized for 3-dimensional cell cultures, e.g., multi-cellular tumor spheroids (MCTS). The SPIM excitation module shapes and deflects the light such that the sample is illuminated by a light sheet perpendicular to the detection path of the microscope. The system is characterized by use of a rectangular capillary for holding (and in an advanced version also by a micro-capillary approach for rotating) the samples, by synchronous adjustment of the illuminating light sheet and the objective lens used for fluorescence detection as well as by adaptation of a microfluidic system for application of fluorescent dyes, pharmaceutical agents or drugs in small quantities. A protocol for working with this system is given, and some technical details are reported. Representative results include (1) measurements of the uptake of a cytostatic drug (doxorubicin) and its partial conversion to a degradation product, (2) redox measurements by use of a genetically encoded glutathione sensor upon addition of an oxidizing agent, and (3) initiation and labeling of cell necrosis upon inhibition of the mitochondrial respiratory chain. Differences and advantages of the present SPIM module in comparison with existing systems are discussed.
Physics, Issue 90, Fluorescence, light sheet, single plane illumination microscopy (SPIM), 3D cell cultures, rectangular capillary, microfluidics, multi-cellular tumor spheroids (MCTS), wide-field microscopy
51993
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Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions
Authors: Chi-Iou Lin, Chun-Yee Lau, Dan Li, Shou-Ching Jaminet.
Institutions: Harvard Medical School.
Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely F-actin-negative membrane projections in endothelial and cancer cells that can be visualized through TM4SF1 (Transmembrane-4-L-six-family-1) immunofluorescence staining. TM4SF1 clusters in 100-300 μm diameter TMED (TM4SF1 enriched microdomains) containing 3 to as many as 14 individual TM4SF1 molecules. TMED are arranged intermittently along nanopodia at a regular spacing of 1 to 3 TMED per μm and firmly anchor nanopodia to matrix. This enables nanopodia to extend more than 100 μm from the leading front or trailing rear of polarized endothelial or tumor cells, and causes membrane residues to be left behind on matrix when the cell moves away. TMED and nanopodia have been overlooked because of their extreme fragility and sensitivity to temperature. Routine washing and fixation disrupt the structure. Nanopodia are preserved by direct fixation in paraformaldehyde (PFA) at 37 °C, followed by brief exposure to 0.01% Triton X-100 before staining. Nanopodia open new vistas in cell biology: they promise to reshape our understanding of how cells sense their environment, detect and identify other cells at a distance, initiate intercellular interactions at close contact, and of the signaling mechanisms involved in movement, proliferation, and cell-cell communications. The methods that are developed for studying TM4SF1-derived nanopodia may be useful for studies of nanopodia that form in other cell types through the agency of classic tetraspanins, notably the ubiquitously expressed CD9, CD81, and CD151.
Cellular Biology, Issue 86, nanopodia, TM4SF1, endothelial cell, tumor cell, F-actin, immunofluorescence staining, tetraspanin
51320
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Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation
Authors: Maciej Kmieciak, Amir Toor, Laura Graham, Harry D. Bear, Masoud H. Manjili.
Institutions: Virginia Commonwealth University- Massey Cancer Center, Virginia Commonwealth University- Massey Cancer Center, Virginia Commonwealth University- Massey Cancer Center.
It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.
Immunology, Issue 47, Adoptive T cell therapy, Breast Cancer, HER-2/neu, common gamma chain cytokines, Bryostatin 1, Ionomycin
2381
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A Sensitive Method to Quantify Senescent Cancer Cells
Authors: Julie Cahu, Brigitte Sola.
Institutions: Université de Caen Basse-Normandie.
Human cells do not indefinitely proliferate. Upon external and/or intrinsic cues, cells might die or enter a stable cell cycle arrest called senescence. Several cellular mechanisms, such as telomere shortening and abnormal expression of mitogenic oncogenes, have been shown to cause senescence. Senescence is not restricted to normal cells; cancer cells have also been reported to senesce. Chemotherapeutical drugs have been shown to induce senescence in cancer cells. However, it remains controversial whether senescence prevents or promotes tumorigenesis. As it might eventually be patient-specific, a rapid and sensitive method to assess senescence in cancer cell will soon be required. To this end, the standard β-galactosidase assay, the currently used method, presents major drawbacks: it is time consuming and not sensitive. We propose here a flow cytometry-based assay to study senescence on live cells. This assay offers the advantage of being rapid, sensitive, and can be coupled to the immunolabeling of various cellular markers.
Cancer Biology, Issue 78, Medicine, Cellular Biology, Anatomy, Physiology, Genetics, Oncology, Tumor Cells, Cultured, Early Detection of Cancer, senescence, cancer, cells, flow cytometry, C12FDG, cell culture, clinical applications
50494
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Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells
Authors: Melissa V. Fernandez, Elizabeth A. Miller, Nina Bhardwaj.
Institutions: New York University School of Medicine, Mount Sinai Medical Center, Mount Sinai Medical Center.
Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1β is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5. Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1β secretion6. Pro-IL-1β protein expression is limited in resting cells; therefore a priming signal is required for IL-1β transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1β secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells (moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin. Monocyte derived DCs are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.
Immunology, Issue 87, NLRP3, inflammasome, IL-1beta, Interleukin-1 beta, dendritic, cell, Nigericin, Toll-Like Receptor 8, TLR8, R848, Monocyte Derived Dendritic Cells
51284
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Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors
Authors: Haiyan S. Li, Stephanie S. Watowich.
Institutions: The University of Texas MD Anderson Cancer Center, The University of Texas Graduate School of Biomedical Sciences.
This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer's patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.
Immunology, Issue 85, hematopoiesis, dendritic cells, Peyer's patch, cytokines, adoptive transfer
51189
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The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice
Authors: Irving C. Allen.
Institutions: Virginia Polytechnic Institute and State University.
The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.
Infection, Issue 86, LPS, Lipopolysaccharide, mouse, pneumonia, gram negative bacteria, inflammation, acute lung inflammation, innate immunity, host pathogen interaction, lung, respiratory disease
51391
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Intralymphatic Immunotherapy and Vaccination in Mice
Authors: Pål Johansen, Thomas M. Kündig.
Institutions: University Hospital Zurich.
Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e. intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 μl of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.
Immunology, Issue 84, Vaccination, Immunization, intralymphatic immunotherapy, Lymph node injection, vaccines, adjuvants, surgery, anesthesia
51031
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Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice: A Model for Immunotherapy Development
Authors: Daniel P. Vang, Gregory T. Wurz, Stephen M. Griffey, Chiao-Jung Kao, Audrey M. Gutierrez, Gregory K. Hanson, Michael Wolf, Michael W. DeGregorio.
Institutions: University of California, Davis, University of California, Davis, Merck KGaA, Darmstadt, Germany.
A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-γ)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.
Medicine, Issue 80, Urinary Bladder, Animals, Genetically Modified, Cancer Vaccines, Immunotherapy, Animal Experimentation, Models, Neoplasms Bladder Cancer, C57BL/6 Mouse, MUC1, Immunotherapy, Preclinical Model
50868
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Strategies for Study of Neuroprotection from Cold-preconditioning
Authors: Heidi M. Mitchell, David M. White, Richard P. Kraig.
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
2192
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Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes
Authors: Fengyang Lei, Rizwanul Haque, Xiaofang Xiong, Jianxun Song.
Institutions: Pennsylvania State University College of Medicine.
Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies. TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs. Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.
Stem Cell Biology, Issue 63, Immunology, T cells, induced pluripotent stem cells, differentiation, Notch signaling, T cell receptor, adoptive cell transfer
3986
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Intra-lymph Node Injection of Biodegradable Polymer Particles
Authors: James I. Andorko, Lisa H. Tostanoski, Eduardo Solano, Maryam Mukhamedova, Christopher M. Jewell.
Institutions: University of Maryland, College Park.
Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these foreign molecules to T and B lymphocytes. Lymph nodes have thus become critical targets for new vaccines and immunotherapies. A recent strategy for targeting these tissues is direct lymph node injection of soluble vaccine components, and clinical trials involving this technique have been promising. Several biomaterial strategies have also been investigated to improve lymph node targeting, for example, tuning particle size for optimal drainage of biomaterial vaccine particles. In this paper we present a new method that combines direct lymph node injection with biodegradable polymer particles that can be laden with antigen, adjuvant, or other vaccine components. In this method polymeric microparticles or nanoparticles are synthesized by a modified double emulsion protocol incorporating lipid stabilizers. Particle properties (e.g. size, cargo loading) are confirmed by laser diffraction and fluorescent microscopy, respectively. Mouse lymph nodes are then identified by peripheral injection of a nontoxic tracer dye that allows visualization of the target injection site and subsequent deposition of polymer particles in lymph nodes. This technique allows direct control over the doses and combinations of biomaterials and vaccine components delivered to lymph nodes and could be harnessed in the development of new biomaterial-based vaccines.
Bioengineering, Issue 83, biomaterial, immunology, microparticle, nanoparticle, vaccine, adjuvant, lymph node, targeting, polymer
50984
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Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury
Authors: Teresa A. Evans, Deborah S. Barkauskas, Jay T. Myers, Alex Y. Huang.
Institutions: Case Western Reserve University, Case Western Reserve University, Case Western Reserve University.
Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury.
Cellular Biology, Issue 93, Intravital, spinal cord crush injury, chimera, microglia, macrophages, dorsal column crush, axonal dieback
52228
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Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages
Authors: Wei Ying, Patali S. Cheruku, Fuller W. Bazer, Stephen H. Safe, Beiyan Zhou.
Institutions: Texas A&M University, Texas A&M University, Texas A&M University.
The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.
Immunology, Issue 76, Cellular Biology, Molecular Biology, Medicine, Genetics, Biomedical Engineering, biology (general), genetics (animal and plant), immunology, life sciences, Life Sciences (General), macrophage polarization, bone marrow derived macrophage, flow cytometry, PCR, animal model
50323
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Collecting And Measuring Wound Exudate Biochemical Mediators In Surgical Wounds
Authors: Brendan Carvalho, David J Clark, David Yeomans, Martin S Angst.
Institutions: Stanford University School of Medicine .
We describe a methodology by which we are able to collect and measure biochemical inflammatory and nociceptive mediators at the surgical wound site. Collecting site-specific biochemical markers is important to understand the relationship between levels in serum and surgical wound, determine any associations between mediator release, pain, analgesic use and other outcomes of interest, and evaluate the effect of systemic and peripheral drug administration on surgical wound biochemistry. This methodology has been applied to healthy women undergoing elective cesarean delivery with spinal anesthesia. We have measured wound exudate and serum mediators at the same time intervals as patient's pain scores and analgesics consumption for up to 48 hours post-cesarean delivery. Using this methodology we have been able to detect various biochemical mediators including nerve growth factor (NGF), prostaglandin E2 (PG-E2) substance P, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNFα, INFγ, G-CSF, GM-CSF, MCP-1 and MIP-1β. Studies applying this human surgical wound bioassay have found no correlations between wound and serum cytokine concentrations or their time-release profile (J Pain. 2008; 9(7):650-7).1 We also documented the utility of the technique to identify drug-mediated changes in wound cytokine content (Anesth Analg 2010; 111:1452-9).2
Medicine, Issue 68, Biochemistry, Anatomy, Physiology, Cytokines, Cesarean Section, Wound Healing, Wounds and Injuries, Surgical Procedures, Operative, Surgical wound, Exudate, cytokines, Substance P, Interleukin 10, Interleukin 6, Nerve growth factor, Prostaglandin E2, Cesarean, Analgesia
50133
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Isolation of Mouse Peritoneal Cavity Cells
Authors: Avijit Ray, Bonnie N. Dittel.
Institutions: Blood Research Institute.
The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of naïve macrophages in the peritoneal cavity makes it a preferred site for the collection of naïve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.
JoVE Immunology, Issue 35, Immune cells, Peritoneal cavity, Macrophage, B cell, B1 cell, isolation procedure
1488
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Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging
Authors: Melanie P. Matheu, Debasish Sen, Michael D Cahalan, Ian Parker.
Institutions: University of California, Irvine (UCI), University of California, Irvine (UCI).
Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and <85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.
Immunology, Issue 17, dendritic cells, mouse, bone marrow, 2-photon imaging, cell culture
773
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.