Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within the brain, ascorbate acts in a neuroprotective and neuromodulatory manner that involves ascorbate cycling between neurons and vicinal astrocytes - a relationship that appears to be crucial for brain ascorbate homeostasis. Additionally, emerging evidence strongly suggests that ascorbate has a greatly expanded role in regulating cellular and systemic iron metabolism than is classically recognized. The increasing recognition of the integral role of ascorbate in normal and deregulated cellular and organismal physiology demands a range of medium-throughput and high-sensitivity analytic techniques that can be executed without the need for highly expensive specialist equipment. Here we provide explicit instructions for a medium-throughput, specific and relatively inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.
21 Related JoVE Articles!
Murine Renal Transplantation Procedure
Institutions: The Ohio State University, The Ohio State University.
Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with > 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.
immunology, Issue 29, mouse, kidney, renal, transplantation, procedure
Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana
Institutions: Institute for Environmental Protection and Research, Regional Agency for Environmental Protection in Emilia-Romagna.
Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia
to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia
) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia
). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2
. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3
. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4
. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.
Chemistry, Issue 62, Artemia franciscana, bioassays, chemical substances, crustaceans, marine environment
Retro-orbital Injection in Adult Zebrafish
Institutions: Children’s Hospital Boston, Harvard Medical School, Dana Farber Cancer Institute.
Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio
), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin
:GFP) cells into adult casper
fish as well as injection of a red fluorescent dye (dextran, Texas Red
) into adult casper
fish. We then visualize successful injections by whole animal fluorescence microscopy.
Cellular Biology, Issue 34, fluorescent dye, kidney marrow cells, vasculature, red blood cells, Zebrafish, injection, retro-orbital injection, transplantation, HSC
Dissection of Organs from the Adult Zebrafish
Institutions: University of Pennsylvania-School of Medicine.
Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease. Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition. Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling. In addition, the bipotential gonad primordium develops into either testis or ovary. This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques. This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.
Developmental Biology, Issue 37, adult, zebrafish, organs, dissection, anatomy
Swimming Performance Assessment in Fishes
Institutions: University of Alberta.
Swimming performance tests of fish have been integral to studies of muscle energetics, swimming mechanics, gas exchange, cardiac physiology, disease, pollution, hypoxia and temperature. This paper describes a flexible protocol to assess fish swimming performance using equipment in which water velocity can be controlled. The protocol involves one to several stepped increases in flow speed that are intended to cause fish to fatigue. Step speeds and their duration can be set to capture swimming abilities of different physiological and ecological relevance. Most frequently step size is set to determine critical swimming velocity (Ucrit
), which is intended to capture maximum sustained swimming ability. Traditionally this test has consisted of approximately ten steps each of 20 min duration. However, steps of shorter duration (e.g. 1 min) are increasingly being utilized to capture acceleration ability or burst swimming performance. Regardless of step size, swimming tests can be repeated over time to gauge individual variation and recovery ability. Endpoints related to swimming such as measures of metabolic rate, fin use, ventilation rate, and of behavior, such as the distance between schooling fish, are often included before, during and after swimming tests. Given the diversity of fish species, the number of unexplored research questions, and the importance of many species to global ecology and economic health, studies of fish swimming performance will remain popular and invaluable for the foreseeable future.
Physiology, Issue 51, fish, swimming, Ucrit, burst, sustained, prolonged, schooling performance
Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements
Institutions: Joint Bioenergy Institute, Lawrence Berkeley National Laboratory.
is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40–50%), hemicellulose (25–30%), and lignin (20–30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas Mäule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana
model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.
Cellular Biology, Issue 87, Xylem, Fibers, Lignin, polysaccharides, Plant cell wall, Mäule staining, Phloroglucinol, Congo red, Toluidine blue O, Calcofluor white, Cell wall staining methods
Habituation and Prepulse Inhibition of Acoustic Startle in Rodents
Institutions: University of Western Ontario.
The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated within a few milliseconds, leading to a whole body flinch in rodents1
. Although startle responses are reflexive responses that can be reliably elicited, they are not stereotypic. They can be modulated by emotions such as fear (fear potentiated startle) and joy (joy attenuated startle), by non-associative learning processes such as habituation and sensitization, and by other sensory stimuli through sensory gating processes (prepulse inhibition), turning startle responses into an excellent tool for assessing emotions, learning, and sensory gating, for review see 2, 3
. The primary pathway mediating startle responses is very short and well described, qualifying startle also as an excellent model for studying the underlying mechanisms for behavioural plasticity on a cellular/molecular level3
We here describe a method for assessing short-term habituation, long-term habituation and prepulse inhibition of acoustic startle responses in rodents. Habituation describes the decrease of the startle response magnitude upon repeated presentation of the same stimulus. Habituation within a testing session is called short-term habituation (STH) and is reversible upon a period of several minutes without stimulation. Habituation between testing sessions is called long-term habituation (LTH)4
. Habituation is stimulus specific5
. Prepulse inhibition is the attenuation of a startle response by a preceding non-startling sensory stimulus6
. The interval between prepulse and startle stimulus can vary from 6 to up to 2000 ms. The prepulse can be any modality, however, acoustic prepulses are the most commonly used.
Habituation is a form of non-associative learning. It can also be viewed as a form of sensory filtering, since it reduces the organisms' response to a non-threatening stimulus. Prepulse inhibition (PPI) was originally developed in human neuropsychiatric research as an operational measure for sensory gating7
. PPI deficits may represent the interface of "psychosis and cognition" as they seem to predict cognitive impairment8-10
. Both habituation and PPI are disrupted in patients suffering from schizophrenia11
, and PPI disruptions have shown to be, at least in some cases, amenable to treatment with mostly atypical antipsychotics12, 13
. However, other mental and neurodegenerative diseases are also accompanied by disruption in habituation and/or PPI, such as autism spectrum disorders (slower habituation), obsessive compulsive disorder, Tourette's syndrome, Huntington's disease, Parkinson's disease, and Alzheimer's Disease (PPI)11, 14, 15
Dopamine induced PPI deficits are a commonly used animal model for the screening of antipsychotic drugs16
, but PPI deficits can also be induced by many other psychomimetic drugs, environmental modifications and surgical procedures.
Neuroscience, Issue 55, Startle responses, rat, mouse, sensory gating, sensory filtering, short-term habituation, long-term habituation, prepulse inhibition
Design and Construction of an Urban Runoff Research Facility
Institutions: Texas A&M University, The Scotts Miracle-Gro Company.
As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use due to increased demand for landscape irrigation. Improper irrigation practices and large rainfall events can result in runoff from urban landscapes which has potential to carry nutrients and sediments into local streams and lakes where they may contribute to eutrophication. A 1,000 m2
facility was constructed which consists of 24 individual 33.6 m2
field plots, each equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated urban landscapes. Runoff volumes from the first and second trials had coefficient of variability (CV) values of 38.2 and 28.7%, respectively. CV values for runoff pH, EC, and Na concentration for both trials were all under 10%. Concentrations of DOC, TDN, DON, PO4
, and Ca2+
had CV values less than 50% in both trials. Overall, the results of testing performed after sod installation at the facility indicated good uniformity between plots for runoff volumes and chemical constituents. The large plot size is sufficient to include much of the natural variability and therefore provides better simulation of urban landscape ecosystems.
Environmental Sciences, Issue 90, urban runoff, landscapes, home lawns, turfgrass, St. Augustinegrass, carbon, nitrogen, phosphorus, sodium
Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis
Institutions: Youngstown State University.
Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5
-derived transposome used in this protocol consists of a linear segment of DNA containing an R6Kγ
replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter
sp. YSU, and randomly incorporates itself into this host’s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli
) strain. The R6Kγ
replication origin allows the plasmid to replicate in pir+ E. coli
strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6Kγ
replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter
sp. YSU or of a wider variety of bacterial strains.
Microbiology, Issue 92, Auxotroph, transposome, transposon, mutagenesis, replica plating, glucose minimal medium, complex medium, Enterobacter
Transplantation of Cells Directly into the Kidney of Adult Zebrafish
Institutions: Massachusetts General Hospital.
Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1
. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2
. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3
. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.
Cellular Biology, Issue 51, zebrafish, kidney, regeneration, transplantation
Assessment of Vascular Function in Patients With Chronic Kidney Disease
Institutions: University of Colorado, Denver, University of Colorado, Boulder.
Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA
) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA
, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA
, aPWV, and vascular endothelial cell protein expression.
Medicine, Issue 88, chronic kidney disease, endothelial cells, flow-mediated dilation, immunofluorescence, oxidative stress, pulse-wave velocity
Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes
Institutions: University of Arkansas for Medical Sciences .
The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-ε improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-α and PKC-ε are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro
model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo
. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-ε mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-ε in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-ε is activated by overexpressing the constitutively active PKC-ε mutant. PKC-ε is inhibited by overexpressing the inactive mutant of PKC-ε. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0
-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0
-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro
Cellular Biology, Issue 71, Biochemistry, Molecular Biology, Genetics, Pharmacology, Physiology, Medicine, Protein, Mitochondrial dysfunction, mitochondria, protein kinase C, renal proximal tubular cells, reactive oxygen species, oxygen consumption, electron transport chain, respiratory complexes, ATP, adenovirus, primary culture, ischemia, cells, flow cytometry
Getting to Compliance in Forced Exercise in Rodents: A Critical Standard to Evaluate Exercise Impact in Aging-related Disorders and Disease
Institutions: Louisiana State University Health Sciences Center.
There is a major increase in the awareness of the positive impact of exercise on improving several disease states with neurobiological basis; these include improving cognitive function and physical performance. As a result, there is an increase in the number of animal studies employing exercise. It is argued that one intrinsic value of forced exercise is that the investigator has control over the factors that can influence the impact of exercise on behavioral outcomes, notably exercise frequency, duration, and intensity of the exercise regimen. However, compliance in forced exercise regimens may be an issue, particularly if potential confounds of employing foot-shock are to be avoided. It is also important to consider that since most cognitive and locomotor impairments strike in the aged individual, determining impact of exercise on these impairments should consider using aged rodents with a highest possible level of compliance to ensure minimal need for test subjects. Here, the pertinent steps and considerations necessary to achieve nearly 100% compliance to treadmill exercise in an aged rodent model will be presented and discussed. Notwithstanding the particular exercise regimen being employed by the investigator, our protocol should be of use to investigators that are particularly interested in the potential impact of forced exercise on aging-related impairments, including aging-related Parkinsonism and Parkinson’s disease.
Behavior, Issue 90, Exercise, locomotor, Parkinson’s disease, aging, treadmill, bradykinesia, Parkinsonism
Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology
Institutions: San Diego State University, University of California San Diego.
Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.
Environmental Sciences, Issue 93, dissolved organic carbon, particulate organic matter, nutrients, DAPI, SYBR, microbial metagenomics, viral metagenomics, marine environment
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
Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays
Institutions: The University of Mississippi.
Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.
Environmental Sciences, Issue 80, Environmental Monitoring, Ecological and Environmental Processes, Environmental Microbiology, Ecology, extracellular enzymes, freshwater microbiology, soil microbiology, microbial activity, enzyme activity
Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization
Institutions: University of Notre Dame.
The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ
hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.
Developmental Biology, Issue 89, animals, vertebrates, fishes, zebrafish, growth and development, morphogenesis, embryonic and fetal development, organogenesis, natural science disciplines, embryo, whole mount in situ hybridization, flat mount, deyolking, imaging
Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney
Institutions: University of Notre Dame.
The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.
Cellular Biology, Issue 90,
zebrafish; kidney; nephron; nephrology; renal; regeneration; proximal tubule; distal tubule; segment; mesonephros; physiology; acute kidney injury (AKI)
A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos
Institutions: University of Notre Dame.
Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ
hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.
Developmental Biology, Issue 93, zebrafish, chemical genetics, chemical screen, in vivo small molecule screen, drug discovery, whole mount in situ hybridization (WISH), high-throughput screening (HTS), high-content screening (HCS)
Single Port Donor Nephrectomy
Institutions: Weill Cornell Medical College of Cornell University, Weill Cornell Medical College of Cornell University.
In 2007, Rane presented the first single port nephrectomy for a small non-functioning kidney at the World Congress of Endourology. Since that time, the use of single port surgery for nephrectomy has expanded to include donor nephrectomy. Over the next two years the technique was adopted for many others types of nephrectomies to include donor nephrectomy. We present our technique for single port donor nephrectomy using the Gelpoint device. We have successfully performed this surgery in over 100 patients and add this experience to our experience of over 1000 laparoscopic nephrectomies. With the proper equipment and technique, single port donor nephrectomy can be performed safely and effectively in the majority of live donors. We have found that our operative times and most importantly our transplant outcomes have not changed significantly with the adoption of the single port donor nephrectomy. We believe that single port donor nephrectomy represents a step forward in the care of living donors.
Medicine, Issue 49, Single Port, Laparoscopic, Donor Nephrectomy, Transplant
Transplantation of Whole Kidney Marrow in Adult Zebrafish
Institutions: Harvard Medical School.
Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (<3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.
Developmental Biology, Issue 2, zebrafish, HSC, stem cells, transplant