Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section1. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).
28 Related JoVE Articles!
Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction
Institutions: RWTH-Aachen University, Kyoto University .
Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2
, immunological responses 3,4
, hemodynamics 5,6
, and small-for-size syndrome 7
. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8
since the first model was described by Lee et al. 9
in 1973, the elimination of the hepatic arterial reconstruction 10
and the introduction of the cuff anastomosis technique by Kamada et al. 11
were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al.
, the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12
and the liver integrity 8,13,14
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo
liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.
Medicine, Issue 73, Biomedical Engineering, Anatomy, Physiology, Immunology, Surgery, liver transplantation, liver, hepatic, partial, orthotopic, split, rat, graft, transplantation, microsurgery, procedure, clinical, technique, artery, arterialization, arterialized, anastomosis, reperfusion, rat, animal model
Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens
Institutions: Institut Pasteur, Paris, France, Institut Pasteur, Paris, France, Institut Pasteur, Paris, France.
are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis
is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri
or Mycobacterium tuberculosis
. The approach makes use of CCF4, a FRET reporter sensitive to β-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, β-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri
as well as multiple mycobacterial strains such as Mycobacterium marinum
, Mycobacterium bovis,
and Mycobacterium tuberculosis
Infection, Issue 76, Infectious Diseases, Immunology, Medicine, Microbiology, Biochemistry, Cellular Biology, Molecular Biology, Pathology, Bacteria, biology (general), life sciences, CCF4-AM, Shigella flexneri, Mycobacterium tuberculosis, vacuolar rupture, fluorescence microscopy, confocal microscopy, pathogens, cell culture
Revealing Dynamic Processes of Materials in Liquids Using Liquid Cell Transmission Electron Microscopy
Institutions: Lawrence Berkeley National Laboratory.
The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted significant interests across the research fields of materials science, physics, chemistry and biology. The key enabling technology is a liquid cell. We fabricate liquid cells with thin viewing windows through a sequential microfabrication process, including silicon nitride membrane deposition, photolithographic patterning, wafer etching, cell bonding, etc. A liquid cell with the dimensions of a regular TEM grid can fit in any standard TEM sample holder. About 100 nanoliters reaction solution is loaded into the reservoirs and about 30 picoliters liquid is drawn into the viewing windows by capillary force. Subsequently, the cell is sealed and loaded into a microscope for in situ imaging. Inside the TEM, the electron beam goes through the thin liquid layer sandwiched between two silicon nitride membranes. Dynamic processes of nanoparticles in liquids, such as nucleation and growth of nanocrystals, diffusion and assembly of nanoparticles, etc., have been imaged in real time with sub-nanometer resolution. We have also applied this method to other research areas, e.g.
, imaging proteins in water. Liquid cell TEM is poised to play a major role in revealing dynamic processes of materials in their working environments. It may also bring high impact in the study of biological processes in their native environment.
Materials Science, Issue 70, Chemical Engineering, Chemistry, Physics, Engineering, Life sciences, Liquid cell, Transmission Electron Microscopy, TEM, In situ TEM, Single nanoparticle trajectory, dynamic imaging, nanocrystals
Creating Dynamic Images of Short-lived Dopamine Fluctuations with lp-ntPET: Dopamine Movies of Cigarette Smoking
Institutions: Yale University, Yale University, Yale University, Yale University, Massachusetts General Hospital, University of California, Irvine.
We describe experimental and statistical steps for creating dopamine movies of the brain from dynamic PET data. The movies represent minute-to-minute fluctuations of dopamine induced by smoking a cigarette. The smoker is imaged during a natural smoking experience while other possible confounding effects (such as head motion, expectation, novelty, or aversion to smoking repeatedly) are minimized.
We present the details of our unique analysis. Conventional methods for PET analysis estimate time-invariant kinetic model parameters which cannot capture short-term fluctuations in neurotransmitter release. Our analysis - yielding a dopamine movie - is based on our work with kinetic models and other decomposition techniques that allow for time-varying parameters 1-7
. This aspect of the analysis - temporal-variation - is key to our work. Because our model is also linear in parameters, it is practical, computationally, to apply at the voxel level. The analysis technique is comprised of five main steps: pre-processing, modeling, statistical comparison, masking and visualization. Preprocessing is applied to the PET data with a unique 'HYPR' spatial filter 8
that reduces spatial noise but preserves critical temporal information. Modeling identifies the time-varying function that best describes the dopamine effect on 11
C-raclopride uptake. The statistical step compares the fit of our (lp-ntPET) model 7
to a conventional model 9
. Masking restricts treatment to those voxels best described by the new model. Visualization maps the dopamine function at each voxel to a color scale and produces a dopamine movie. Interim results and sample dopamine movies of cigarette smoking are presented.
Behavior, Issue 78, Neuroscience, Neurobiology, Molecular Biology, Biomedical Engineering, Medicine, Anatomy, Physiology, Image Processing, Computer-Assisted, Receptors, Dopamine, Dopamine, Functional Neuroimaging, Binding, Competitive, mathematical modeling (systems analysis), Neurotransmission, transient, dopamine release, PET, modeling, linear, time-invariant, smoking, F-test, ventral-striatum, clinical techniques
Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa
Institutions: Université J. Fourier.
Effectively determining masses of proteins is critical to many biological studies (e.g.
for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. 13
Electrospray ionization (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra.
Here we present an accessible approach for analyzing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e.
2,5-dihydroxybenzoic acid and α-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analyzing intact proteins larger than 100 kDa by MALDI-MS.
Chemistry, Issue 79, Chemistry Techniques, Analytical, Mass Spectrometry, Analytic Sample Preparation Methods, biochemistry, Analysis of intact proteins, mass spectrometry, matrix-assisted laser desorption ionization, time of flight, sample preparation
High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function
Institutions: Royal Victoria Hospital, McGill University Health Centre Research Institute.
This article focuses on whole-mount in situ
hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro
transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.
Neuroscience, Issue 80, Blood Cells, Endoderm, Motor Neurons, life sciences, animal models in situ hybridization, morpholino knockdown, progranulin, neuromast, proprotein convertase, anti-sense transcripts, intermediate cell mass, pronephric duct, somites
A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent
Institutions: University of Maine, Orono, University of Maine, Orono.
Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g.
by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5
. Mast cell granule component and the allergic mediator β-hexosaminidase, which is released linearly in tandem with histamine from mast cells6
, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1
. Originally published by Naal et al.1
, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11
, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2
. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using ε280
= 4,200 L/M/cm)12
. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.
Immunology, Issue 81, mast cell, basophil, degranulation, RBL-2H3, triclosan, irgasan, antibacterial, β-hexosaminidase, allergy, Asthma, toxicants, ionophore, antigen, fluorescence, microplate, UV-Vis
An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei
Institutions: University Hospital Heidelberg, Research Center Borstel.
Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium
, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e.
aerosol and mosquito bite, respectively.
Infectious Diseases, Issue 84, coinfection, mouse, Tuberculosis, Malaria, Plasmodium berghei, Mycobacterium tuberculosis, natural transmission
High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities
Institutions: Colorado State University, Oak Ridge National Laboratory, University of Colorado.
Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be assimilated for energy and nutrients. Measuring soil microbial enzyme activity is crucial in understanding soil ecosystem functional dynamics. The general concept of the fluorescence enzyme assay is that synthetic C-, N-, or P-rich substrates bound with a fluorescent dye are added to soil samples. When intact, the labeled substrates do not fluoresce. Enzyme activity is measured as the increase in fluorescence as the fluorescent dyes are cleaved from their substrates, which allows them to fluoresce. Enzyme measurements can be expressed in units of molarity or activity. To perform this assay, soil slurries are prepared by combining soil with a pH buffer. The pH buffer (typically a 50 mM sodium acetate or 50 mM Tris buffer), is chosen for the buffer's particular acid dissociation constant (pKa) to best match the soil sample pH. The soil slurries are inoculated with a nonlimiting amount of fluorescently labeled (i.e.
C-, N-, or P-rich) substrate. Using soil slurries in the assay serves to minimize limitations on enzyme and substrate diffusion. Therefore, this assay controls for differences in substrate limitation, diffusion rates, and soil pH conditions; thus detecting potential enzyme activity rates as a function of the difference in enzyme concentrations (per sample).
Fluorescence enzyme assays are typically more sensitive than spectrophotometric (i.e.
colorimetric) assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light; so caution is required when handling fluorescent substrates. Likewise, this method only assesses potential enzyme activities under laboratory conditions when substrates are not limiting. Caution should be used when interpreting the data representing cross-site comparisons with differing temperatures or soil types, as in situ
soil type and temperature can influence enzyme kinetics.
Environmental Sciences, Issue 81, Ecological and Environmental Phenomena, Environment, Biochemistry, Environmental Microbiology, Soil Microbiology, Ecology, Eukaryota, Archaea, Bacteria, Soil extracellular enzyme activities (EEAs), fluorometric enzyme assays, substrate degradation, 4-methylumbelliferone (MUB), 7-amino-4-methylcoumarin (MUC), enzyme temperature kinetics, soil
A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening
Institutions: Université de Lille.
Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis
) and to search for novel chemicals that could become TB drugs. One approach is to set up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb
within fluorescently labeled host cells using automated confocal microscopy. This in vitro
assay allows an image based quantification of the colonization process of Mtb
into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb
mutant-libraries. The images are then processed for multiparametric analysis, which provides read out inferring on the pathogenesis of Mtb
within host cells.
Infection, Issue 83, Mycobacterium tuberculosis, High-content/High-throughput screening, chemogenomics, Drug Discovery, siRNA library, automated confocal microscopy, image-based analysis
Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray
Institutions: Akonni Biosystems, Inc..
Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis
(MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.
Immunology, Issue 86, MDR-TB, gel element microarray, closed amplicon, drug resistance, rifampin, isoniazid, streptomycin, ethambutol
An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons
Institutions: Boston Children's Hospital, Harvard Medical School.
Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and subsequently digests this material, commonly through lysosomal degradation. Microglia are the resident immune cells of the central nervous system (CNS) whose phagocytic function has been described in a broad range of conditions from neurodegenerative disease (e.g.
, beta-amyloid clearance in Alzheimer’s disease) to development of the healthy brain (e.g.,
. The following protocol is an engulfment assay developed to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing mouse retinogeniculate system7
. While this assay was used to assess microglia function in this particular context, a similar approach may be used to assess other phagocytes throughout the brain (e.g.,
astrocytes) and the rest of the body (e.g.
, peripheral macrophages) as well as other contexts in which synaptic remodeling occurs (e.g.
Neuroscience, Issue 88, Central Nervous System (CNS), Engulfment, Phagocytosis, Microglia, Synapse, Anterograde Tracing, Presynaptic Input, Retinogeniculate System
Growth of Mycobacterium tuberculosis Biofilms
Institutions: University of Pittsburgh, University of Pittsburgh.
, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis
is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1
, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4
. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6
. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7
In a series of recent papers we established that M. tuberculosis
and Mycobacterium smegmatis
have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10
. M. tuberculosis,
however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9
. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis
is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis
biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis
, with deletion in the two loci, panCD
that are critical for in vivo
growth of the pathogen 9
. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.
Immunology, Issue 60, Mycobacterium tuberculosis, tuberculosis, drug tolerance, biofilms
Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies
Institutions: University of Nebraska-Lincoln, University of Nebraska-Lincoln.
is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis
has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis
cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1
harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 °C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1
H NMR and two-dimensional (2D) 1
C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.
Infection, Issue 67, Mycobacterium tuberculosis, NMR, Metabolomics, homogenizer, lysis, cell extracts, sample preparation
Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format
Institutions: Mayo Clinic .
The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioMérieux, Hazelwood, MO). The agar proportion method, while considered the “gold” standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is ≥1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.
Immunology, Issue 52, Mycobacterium tuberculosis, MIC, antimicrobial susceptibility testing, first and second line drugs, microtiter plate, broth dilution
Aseptic Laboratory Techniques: Plating Methods
Institutions: University of California, Los Angeles .
Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories
(BMBL) as well as Material Safety Data Sheets
(MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection
(ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to:
● Perform plating procedures without contaminating media.
● Isolate single bacterial colonies by the streak-plating method.
● Use pour-plating and spread-plating methods to determine the concentration of bacteria.
● Perform soft agar overlays when working with phage.
● Transfer bacterial cells from one plate to another using the replica-plating procedure.
● Given an experimental task, select the appropriate plating method.
Basic Protocols, Issue 63, Streak plates, pour plates, soft agar overlays, spread plates, replica plates, bacteria, colonies, phage, plaques, dilutions
Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice
Institutions: Charles River, Charles River, University of Washington.
Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.
Immunology, Issue 55, rat, mouse, endoparasite, ectoparasite, diagnostics, mites, pinworm, helminths, protozoa, health monitoring
Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells
Institutions: Northwestern University.
Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2
. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7
. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8
. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7
. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9
. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10
. This protein cannot fold properly at 39°C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31°C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11
. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.
Cellular Biology, Issue 51, Epithelial cells, cell polarity, microinjection, basolateral sorting, MDCK
Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice
Institutions: Cornell University.
Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm-/-
consistently develop thymic lymphoma early in life 1,2
, and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols 1,3
. Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain 1,4
. We have successfully established several T-cell lines using this protocol and inbred strains ofAtm-/-
mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year.
Immunology, Issue 50, mouse, thymic lymphoma, Atm, p53, T-cell lines
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
Intravitreous Injection for Establishing Ocular Diseases Model
Institutions: The University of Hong Kong - HKU.
Intravitreous injection is a widely used technique in visual sciences research. It can be used to establish animal models with ocular diseases or as direct application of local treatment. This video introduces how to use simple and inexpensive tools to finish the intravitreous injection procedure. Use of a 1 ml syringe, instead of a hemilton syringe, is used. Practical tips for how to make appropriate injection needles using glass pipettes with perfect tips, and how to easily connect the syringe needle with the glass pipette tightly together, are given.
To conduct a good intravitreous injection, there are three aspects to be observed: 1) injection site should not disrupt retina structure; 2) bleeding should be avoided to reduce the risk of infection; 3) lens should be untouched to avoid traumatic cataract. In brief, the most important point is to reduce the interruption of normal ocular structure. To avoid interruption of retina, the superior nasal region of rat eye was chosen. Also, the puncture point of the needle was at the par planar, which was about 1.5 mm from the limbal region of the rat eye. A small amount of vitreous is gently pushed out through the puncture hole to reduce the intraocular pressure before injection. With the 45° injection angle, it is less likely to cause traumatic cataract in the rat eye, thus avoiding related complications and influence from lenticular factors. In this operation, there was no cutting of the conjunctiva and ocular muscle, no bleeding. With quick and minor injury, a successful intravitreous injection can be done in minutes.
The injection set outlined in this particular protocol is specific for intravitreous injection. However, the methods and materials presented here can also be used for other injection procedures in drug delivery to the brain, spinal cord or other organs in small mammals.
Neuroscience, Issue 8, eye, injection, rat
The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis
Institutions: The Warren Alpert Medical School of Brown University, Universidad Peruana Cayetano Heredia, Johns Hopkins Bloomberg School of Public Health, Imperial College London .
Patients with active pulmonary tuberculosis (TB) infect 10-15 other persons per year, making diagnosing active TB essential to both curing the patient and preventing new infections. Furthermore, the emergence of multidrug resistant tuberculosis (MDRTB) means that detection of drug resistance is necessary for stopping the spread of drug-resistant strains. The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of TB and MDRTB. The MODS assay is based on three principles: 1) mycobacterium tuberculosis (MTB) grows faster in liquid media than on solid media 2) microscopic MTB growth can be detected earlier in liquid media than waiting for the macroscopic appearance of colonies on solid media, and that growth is characteristic of MTB, allowing it to be distinguished from atypical mycobacteria or fungal or bacterial contamination 3) the drugs isoniazid and rifampicin can be incorporated into the MODS assay to allow for simultaneous direct detection of MDRTB, obviating the need for subculture to perform an indirect drug susceptibility test. Competing current diagnostics are hampered by low sensitivity with sputum smear, long delays until diagnosis with solid media culture, prohibitively high cost with existing liquid media culture methods, and the need to do subculture for indirect drug susceptibility testing to detect MDRTB. In contrast, the non-proprietary MODS method has a high sensitivity for TB and MDRTB, is a relatively rapid culture method, provides simultaneous drug susceptibility testing for MDRTB, and is accessible to resource-limited settings at just under $3 for testing for TB and MDRTB.
Microbiology, Issue 18, tuberculosis, TB, multidrug resistant tuberculosis, MDRTB, culture, diagnostic
Phase Contrast and Differential Interference Contrast (DIC) Microscopy
Institutions: University of Texas Health Science Center at San Antonio (UTHSCSA).
Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the phase halos typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.
Basic protocols, Issue 18, Current Protocols Wiley, Microscopy, Phase Contrast, Difference Interference Contrast
Reaggregate Thymus Cultures
Institutions: University of Birmingham .
Stromal cells within lymphoid tissues are organized into three-dimensional structures that provide a scaffold that is thought to control the migration and development of haemopoeitic cells. Importantly, the maintenance of this three-dimensional organization appears to be critical for normal stromal cell function, with two-dimensional monolayer cultures often being shown to be capable of supporting only individual fragments of lymphoid tissue function. In the thymus, complex networks of cortical and medullary epithelial cells act as a framework that controls the recruitment, proliferation, differentiation and survival of lymphoid progenitors as they undergo the multi-stage process of intrathymic T-cell development. Understanding the functional role of individual stromal compartments in the thymus is essential in determining how the thymus imposes self/non-self discrimination. Here we describe a technique in which we exploit the plasticity of fetal tissues to re-associate into intact three-dimensional structures in vitro
, following their enzymatic disaggregation. The dissociation of fetal thymus lobes into heterogeneous cellular mixtures, followed by their separation into individual cellular components, is then combined with the in vitro
re-association of these desired cell types into three-dimensional reaggregate structures at defined ratios, thereby providing an opportunity to investigate particular aspects of T-cell development under defined cellular conditions. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).
Immunology, Issue 18, Springer Protocols, Thymus, 2-dGuo, Thymus Organ Cultures, Immune Tolerance, Positive and Negative Selection, Lymphoid Development
Preparation of 2-dGuo-Treated Thymus Organ Cultures
Institutions: University of Birmingham .
In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo
can limit analysis of individual cellular components and particular stages of development. In vitro
culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro
. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro
with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).
Immunology, Issue 18, Springer Protocols, Thymus, 2-dGuo, Thymus Organ Cultures, Immune Tolerance, Positive and Negative Selection, Lymphoid Development
Monitoring Plant Hormones During Stress Responses
Institutions: University of Texas.
Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. Among the most severe stresses are insect herbivory, pathogen infection, and drought stress. For each of these stresses a specific set of hormones and/or combinations thereof are known to fine-tune the responses, thereby ensuring the plant's survival. The major hormones involved in the regulation of these responses are jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). To better understand the role of individual hormones as well as their potential interaction during these responses it is necessary to monitor changes in their abundance in a temporal as well as in a spatial fashion. For the easy, sensitive, and reproducible quantification of these and other signaling compounds we developed a method based on vapor phase extraction and gas chromatography/mass spectrometry (GC/MS) analysis (1, 2, 3, 4). After extracting these compounds from the plant tissue by acidic aqueous 1-propanol mixed with dichloromethane the carboxylic acid-containing compounds are methylated, volatilized under heat, and collected on a polymeric absorbent. After elution into a sample vial the analytes are separated by gas chromatography and detected by chemical ionization mass spectrometry. The use of appropriate internal standards then allows for the simple quantification by relating the peak areas of analyte and internal standard.
Plant Biology, Issue 28, Jasmonic acid, salicylic acid, abscisic acid, plant hormones, GC/MS, vapor phase extraction
Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of Obliterative Airway Disease
Institutions: University Heart Center Hamburg, University Hospital Hamburg, Stanford University School of Medicine.
Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the pathophysiology, treatment and prevention of OAD, different animal models of tracheal transplantation in rodents have been developed (1-7). Here, we use two established models of trachea transplantation, the heterotopic and orthotopic model and demonstrate their advantages and limitations.
For the heterotopic model, the donor trachea is wrapped into the greater omentum of the recipient, whereas the donor trachea is anastomosed by end-to-end anastomosis in the orthotopic model.
In both models, the development of obliterative lesions histological similar to clinical OAD has been demonstrated (1-7).
This video shows how to perform both, the heterotopic as well as the orthotopic tracheal transplantation technique in mice, and compares the time course of OAD development in both models using histology.
Immunology, Issue 35, orthotopic tracheal transplantation, heterotopic tracheal transplantation, obliterative airway disease, mice, luminal obliteration, histology
Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device
Institutions: University of California, Irvine (UCI), University of California, Irvine (UCI), University of California, Irvine (UCI).
In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4°C for up to a week before the dissociation is performed. However, there will be a drop in cell viability. Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37°C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.
Neuroscience, Issue 8, Biomedical Engineering, Neurons, Axons, Axonal Regeneration, Neuronal Culture, Cell Culture