Journal of Biological Methods (JBM)
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Generation of axolotl hematopoietic chimeras
No full textWound repair is an extremely complex process that requires precise coordination between various cell types including immune cells. Unfortunately, in mammals this usually results in scar formation instead of restoration of the original fully functional tissue, otherwise known as regeneration. Various animal models like frogs and salamanders are currently being studied to determine the intracellular and intercellular pathways, controlled by gene expression, that elicit cell proliferation, differentiation, and migration of cells during regenerative healing. Now, the necessary genetic tools to map regenerative pathways are becoming available for the axolotl salamander, thus allowing comparative studies between scarring and regeneration. Here, we describe in detail three methods to produce axolotl hematopoietic cell-tagged chimeras for the study of hematopoiesis and regeneration
A simple, effective and inexpensive method to highlight antigen-antibody reaction based on liquid-semisolid phase
No full textIdentification of the immunological antigen-antibody reaction performed either in liquid phase or in agarose have several limits: antigen excess, low sensitivity, low speed reaction. A new method is described for the antigen-antibody reaction, easy, simple to perform and inexpensive. It is based on a reaction where the specific antibody is mixed with a semi-solid gel on a transparent surface and the antigen to be identified is inoculated into the gel. In this method, an immune precipitate can be shown in just few minutes, obtaining a good sensitivity compared with manual methods such as the Ouchterlony technique and other similar methods. By using albumin, IgG, RBP, transferrin, alpha-2-macroglobulin as antigen, the immune precipitate is formed within 10 min at 2–8 microgram/ml and, after 30 min, at 1–4 microgram/ml may be detected. The display of the immune precipitate is favored by oblique observation on a black background with a perpendicular light illuminating the gel from the bottom upwards. This method also allows a good control of antigen excess, a problem that is instead found in immunoturbidimetric reactions. The method here described has also been tested in serum and urine, and then it can be advantageously used as qualitative or semi-quantitative immunological test in research or diagnostic. Moreover, it could be particularly useful in low resources geographical areas, where an immunoassay that requires the use of dedicated equipment is not economically sustainable. The authors have waived the patent rights to allow the free development of new tests based on this method
Scientific methods
No full text“工欲善其事,必先利其器” --‘If a workman wishes to do a good job, he must first sharpen his tools.’ This ancient adage from Confucius maintains its relevance even now in the information age where the potential applications of new technology are limited only by the imagination. Historically, the development and dissemination of novel techniques & methods has served as a catalyst for scientific progress by re-defining what is possible within the laboratory. It is worth recounting that the breakthroughs in genetic engineering that led to the production of the first recombinant protein and the birth of biotechnology occurred within a decade of the discovery of the first restriction enzyme. Once again, we are on the cusp of a paradigm shift in the biological sciences following the discovery of yet another mechanism of prokaryotic immunity; the CRISPR/Cas system. A series of landmark publications involving a single enzyme in this system have seen it emerge as an incredibly versatile and powerful tool capable of manipulating the transcriptome, epigenome and even the genome itself. The rate at which such technology can be adapted and repurposed by the scientific community is a testament to the power of open access resources, and their capacity to facilitate and accelerate the exchange of ideas. Given the importance of reliable techniques and methodologies in advancing scientific research, we are pleased to announce the launch of the Journal of Biological Methods (JBM, ISSN 2326-9901), a peer-reviewed open access journal dedicated to the publication of innovative, cutting-edge methods and techniques across the spectrum of life sciences
A stepwise procedure to test contractility and susceptibility to injury for the rodent quadriceps muscle
No full textIn patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers can also be used in measuring muscle injury, such as increased creatine kinase levels in the blood, but these are not always correlated with loss in muscle function (i.e. loss of force production). This is even true of histological findings from animals, which provide a “direct measure” of damage, but do not account for loss of function. The most comprehensive measure of the overall health of the muscle is contractile force. To date, animal models testing contractile force have been limited to the muscle groups moving the ankle. Here we describe an in vivo animal model for the quadriceps, with abilities to measure torque, produce a reliable muscle injury, and follow muscle recovery within the same animal over time. We also describe a second model used for direct measurement of force from an isolated quadriceps muscle in situ.
Automated high-throughput measurement of body movements and cardiac activity of Xenopus tropicalis tadpoles
No full textXenopus tadpoles are an emerging model for developmental, genetic and behavioral studies. A small size, optical accessibility of most of their organs, together with a close genetic and structural relationship to humans make them a convenient experimental model. However, there is only a limited toolset available to measure behavior and organ function of these animals at medium or high-throughput. Herein, we describe an imaging-based platform to quantify body and autonomic movements of Xenopus tropicalis tadpoles of advanced developmental stages. Animals alternate periods of quiescence and locomotor movements and display buccal pumping for oxygen uptake from water and rhythmic cardiac movements. We imaged up to 24 animals in parallel and automatically tracked and quantified their movements by using image analysis software. Animal trajectories, moved distances, activity time, buccal pumping rates and heart beat rates were calculated and used to characterize the effects of test compounds. We evaluated the effects of propranolol and atropine, observing a dose-dependent bradycardia and tachycardia, respectively. This imaging and analysis platform is a simple, cost-effective high-throughput in vivo assay system for genetic, toxicological or pharmacological characterizations
Proof of concept for a novel functional screening system for plant sucrose effluxers
No full textMembrane transporters play pivotal roles in facilitating sucrose transport in plants and their activities have been shown to impact plant growth rates and crop yield. In contrast to the well-defined mechanism of sucrose influx across plasma membranes, less is known about sucrose efflux mechanisms and the membrane proteins supporting this function. A major impediment blocking progress in this key area of plant science is the absence of a functional screening system for genes encoding sucrose effluxers. Here we report a novel yeast system for screening sucrose effluxers based on sucrose release from yeast cells genetically modified to synthesize, but not to metabolize, sucrose. Inhibiting sucrose metabolism was achieved using yeast strains, SEY 6210 and YSL4-6, carrying mutations in genes encoding invertase and maltase, respectively. Genes encoding essential components of sucrose biosynthesis, sucrose phosphate synthase (SPS) and sucrose phosphate phosphatase (SPP), were used to transform the two yeast hosts to make strains SuPy (from SEY6210) and Ysu (from YSL4-6). Cultures of SuPy15 cells were found to be capable of synthesizing sucrose when supplied with various compounds as the sole carbon source, including non-fermentable sugars and non-sugar substrates. A proof of concept of the screening system was demonstrated by transforming SuPy15 with sucrose transporter genes known to encode plasma membrane proteins that mediate sucrose efflux. The robustness of the yeast SuPy15 system as a novel platform to screen putative plant sucrose effluxers is discussed
Anomalous Subdiffusive Measurements by Fluorescence Correlations Spectroscopy and Simulations of Translational Diffusive Behavior in Live Cells
No full textUsing a combination of optical experiments and computer simulations, we found that live cells act as traps to produce anomalous subdiffusive translational motion of molecules exemplified by the glucocorticoid receptor α. The glucocorticoid receptor α was expressed as a fusion protein with the green fluorescent protein in living mammalian U2OS cells. The measurements were carried out by fluorescence correlation spectroscopy. The glucocorticoid receptor α was either a homodimer or a monomer in the nucleoplasm depending on the presence or absence of the stimulus dexamethasone. Our simulations showed that the experimentally measured rates of translational diffusion could be attributed to spatial and temporal traps. Thus, traps acted by spatial and temporal heterogeneity and randomness, respectively. As we prove here for the first time, anomalous translational diffusion caused by spatial randomness was different from anomalous translational diffusion caused by heterogeneous temporal randomness. For this purpose, we explored two classes of transformations in the time domain for which the assumption that they are stable and have probability density functions was satisfied. The first one was the Inverse Gamma distribution and the second class was a stable Levy distribution. The spatial exponent α that was extracted from time series expressed scale invariance in the space domain. The time exponent γ measured limited time scaling of the embedding complex dynamics. Hence, both exponents must not be mixed up by a single exponent. Fluorescence fluctuation spectroscopy (FCS) is the method of choice for measuring the exponents of anomalous subdiffusive translational motion of molecules in live cells
Advanced methods of microscope control using μManager software
No full textµManager is an open-source, cross-platform desktop application, to control a wide variety of motorized microscopes, scientific cameras, stages, illuminators, and other microscope accessories. Since its inception in 2005, µManager has grown to support a wide range of microscopy hardware and is now used by thousands of researchers around the world. The application provides a mature graphical user interface and offers open programming interfaces to facilitate plugins and scripts. Here, we present a guide to using some of the recently added advanced µManager features, including hardware synchronization, simultaneous use of multiple cameras, projection of patterned light onto a specimen, live slide mapping, imaging with multi-well plates, particle localization and tracking, and high-speed imaging
A noninvasive assay for monitoring renal allograft status
No full textTransplant rejection is a serious complication, sometimes threatening life of the patient. Although recent development of the new generation of immunosuppressive drugs reduced the incidence of acute rejection in kidney transplantation, the absence of noninvasive biomarkers of the rejection does not allow often the optimization of a prompt antirejection therapy. Serum creatinine is the most widely used marker for allograft function, however, it is not sensitive and specific enough to detect acute rejection. Other biomarkers are even less valuable for this purpose. Histological examination of renal allograft biopsy still remains the golden standard for diagnosing acute renal allograft rejection. Therefore, there is a high demand for reliable biomarkers for noninvasive monitoring of renal allograft status. Examination of urine in renal transplant recipients provides a logical and readily accessible approach for this monitoring. The high potency biomarkers for kidney allograft monitoring are fragments of DNA in recipient urine that originated from renal allograft cells. Because of the difference in the genetic origin these DNA can be distinguished from recipient DNA. Quantitative analysis of donor’s DNA, derived from cells of renal allograft, in recipient’s urine might be a reliable predictive tool for the kidney transplant rejection. We developed an assay to quantitate donor DNA content in recipient urine. Application of the technique—coamplification at lower denaturation temperature-PCR (COLD-PCR) increased the abundance of donor DNA that usually presents in recipient urine in quantities that are out of the detection range. This assay has a potential for routine application in clinical practice after statistical validation and additional modifications.
A stepwise procedure for isolation of murine bone marrow and generation of dendritic cells
No full textBone marrow derived Dendritic cells (BMDCs) are routinely employed in cell based assays to evaluate immunomodulatory and anti-inflammatory activities. Hence, simplified, stepwise, defined and standardized methods are required for isolation of bone marrow cells from mice, propagating them in presence of growth factors and obtaining high and reproducible yields of BMDCs. Here, we describe a detailed, stepwise protocol with pictorial representation to isolate bone marrow from mouse femur and development of dendritic cells. Mouse bone marrow cells are cultured in presence of granulocyte-macrophage colony stimulating factor (GM-CSF) for 6 days to generate BMDCs