Journal of Biological Methods (JBM)
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163 research outputs found
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Removing nucleic acids from nucleoid-Associated proteins purified by affinity column
No full textIn bacteria, DNA is tightly compacted in a supercoiled organization, which is mediated in part by nucleoid-associated proteins (NAPs). NAPs are well characterized for their ability to bind nucleic acids and for their involvement in gene regulation. A method commonly used to study protein-nucleic acid interactions involves immunoprecipitation of the protein of interest which is subsequently incubated with nucleic acids. A common cause of artifact is due to nucleic acids that remains bound to the protein of interest during the whole purification process. We developed an optimized method for the purification of tagged NAPs on affinity columns. The combination of three known methods allows removal of most of the nucleic acids bound to proteins during the purification process. This protocol is designed to improve the quality and specificity of results of in vitro experiments involving nucleic acid binding tests on purified NAPs. It can be used for in vitro studies of other RNA/DNA binding proteins
ChIP and Chips: Introducing the WormPharm for correlative studies employing pharmacology and genome-wide analyses in C. elegans
No full textWe present the WormPharm, an automated microfluidic platform that utilizes an axenic medium to culture C. elegans. The WormPharm is capable of sustaining C elegans for extended periods, while recording worm development and growth with high temporal resolution ranging from seconds to minutes over several days to months. We demonstrate the utility of the device to monitor C. elegans growth in the presence of varying doses of nicotine and alcohol. Furthermore, we show that C. elegans cultured in the WormPharm are amendable for high-throughput genomic assays, i.e. chromatin-immunoprecipitation followed by next generation sequencing, and confirm that nematodes grown in monoxenic and axenic cultures exhibit genetic modifications that correlate with observed phenotypes. The WormPharm is a powerful tool for analyzing the effects of chemical, nutritional and environmental variations on organism level responses in conjunction with genome-wide changes in C. elegans
A comparison of strategies for immortalizing mouse embryonic fibroblasts
No full textThe genetically amenable mouse model has led to a large collection of genetically defined lines from which mouse embryonic fibroblasts (MEFs) have been derived. Despite their widespread use, MEFs are time consuming to generate and have a limited lifespan. Immortalizing primary MEFs with the desired genetic manipulations greatly reduces culture maintenance time, enables the generation of near limitless amounts of protein lysate, and facilitates biological replicates during experimentation. In this work, we have evaluated several approaches for MEF immortalization. When cultivated at 3% O2, some primary MEF lines could be proliferated for > 40 passages with a median doubling rate of 45 ± 55 h (n = 8). However, serial passaging at 3% O2 achieved spontaneous immortalization with varying success. If cultures seemed to be reaching their Hayflick limit when cultivated at 3% O2, supplementing the culture media with 5 µM ROCK inhibitor Y-27632 helped to extend proliferation and achieve spontaneous immortalization. MEFs immortalized via SV40 Ta infection reliably produced cell lines with a median doubling rate of 25 ± 9 h (n = 9) and viability greater than 90%. In addition to a discussion of the characteristics of cell lines generated with various immortalization strategies, pros and cons of each strategy are included as are recommendations for generating immortalized MEFs
High-contrast enzymatic immunohistochemistry of pigmented tissues
No full textHistorically, standard enzyme immunohistochemistry has been accomplished with brown (DAB, diaminobenzidine) substrate. This can become problematic in pigmented tissues, such as the retina, where brown pigment of retinal pigmented epithelial (RPE) cells can be easily confounded with brown substrate. Although immunofluorescence detection methods can overcome this challenge, fluorescence may fade over a period of weeks, while enzyme substrates allow for more long-lasting, archival results. In this report, we will describe a high-contrast enzyme immunohistochemistry method ideal for pigmented tissues that utilizes purple (VIP) substrate. We compared brown (DAB) and purple (VIP) substrates in enzyme immunohistochemistry experiments using human retina (paraffin sections) and monkey retinal pigmented epithelial cells (frozen sections), both containing brown pigmented cells. We compared substrates using several primary antibodies against markers that can be detected in the retina, including GFAP, VEGF, CD147 (EMMPRIN), RHO (rhodopsin) and PAX6. Methyl green was used as a counterstain for paraffin sections. A side-by-side comparison between DAB and VIP immunohistochemistry showed excellent contrast between pigmented cells and the purple VIP substrate in both human retinal tissue and monkey pigmented epithelial cells for all of the markers tested. This was a marked improvement over DAB staining in pigmented cells and tissues. For both paraffin sections and frozen sections of pigmented tissues, purple VIP substrate is an excellent alternative to brown DAB substrate and non-permanent immunofluorescence methods
A three-dimensional co-culture system to investigate macrophage-dependent tumor cell invasion
No full textMacrophages infiltrate cancers and promote progression to invasion and metastasis. To directly examine tumor-associated macrophages (TAMs) and tumor cells interacting and co-migrating in a three-dimensional (3D) environment, we have developed a co-culture model that uses a PyVmT mouse mammary tumor-derived cell line and mouse bone marrow-derived macrophages (BMM). The Py8119 cell line was cloned from a spontaneous mammary tumor in a Tg(MMTV:LTR-PyVmT) C57Bl/6 mouse and these cells form 3-dimensional (3D) spheroids under conditions of low adhesion. Co-cultured BMM infiltrate the Py8119 mammospheres and embedding of the infiltrated mammospheres in Matrigel leads to subsequent invasion of both cell types into the surrounding matrix. This physiologically relevant co-culture model enables examination of two critical steps in the promotion of invasion and metastasis by BMM: 1) macrophage infiltration into the mammosphere and, 2) subsequent invasion of macrophages and tumor cells into the matrix. Our methodology allows for quantification of BMM infiltration rates into Py8119 mammospheres and demonstrates that subsequent tumor cell invasion is dependent upon the presence of infiltrated macrophages. This method is also effective for screening macrophage motility inhibitors. Thus, we have developed a robust 3D in vitro co-culture assay that demonstrates a central role for macrophage motility in the promotion of tumor cell invasion
Assessing zygosity in progeny of transgenic plants: current methods and perspectives
No full textHomozygosity is highly desirable in transgenic plants research to ensure the stable integration and inheritance of transgene(s). Simple, reliable and high-throughput techniques to detect the zygosity of transgenic events in plants are invaluable tools for biotechnology and plant breeding companies. Currently, a number of basic techniques are being used to determine the zygosity of transgenic plants in T1 generation. For successful application of any technique, precision and simplicity of approach combined with the power of resolution are important parameters. On the basis of simplicity, resolution and cost involved, the available techniques have been classified into three major classes which are conventional methods, current methods and next generation methods. Conventional methods include antibiotic marker-based selection and the highly labour intensive Southern blot analysis. In contrast, methods such as real time PCR, TAIL PCR and competitive PCR are not only cost effective but rapid as well. Moreover, methods such as NGS, Digital PCR and Loop-mediated isothermal amplification also provide cost effective, fast and not so labour-intensive provide substitute of current methods. In this review we have attempted to compare and contrast all the available efficient methods to distinguish the homozygous plant in progeny of transgenics. This review also provides plenty of information regarding the various techniques available for determining zygosity in plants so as to permit the scientists to make informed choices of techniques that best suit their analyses. More importantly, detection and subsequent selection of homozygous individuals is central for facilitating the movement of transgenic plant from the laboratory to the field
Direct lentivirus injection for fast and efficient gene transfer into brown and beige adipose tissue
No full textBrown adipose tissue is a special type of fat contributing to energy expenditure in human newborns and adults. Moreover, subcutaneous white adipose tissue has a high capacity to adapt an energy-consuming, brown-like/beige phenotype. Here, we developed an easy to handle and fast to accomplish method to efficiently transfer genes into brown and beige fat pads in vivo. Lentiviral vectors are directly injected into the target fat pad of anaesthetized mice through a small incision using a modified, small needle connected to a microsyringe, which is well suited for infiltration of adipose tissues. Expression of the target gene can be detected in brown/beige fat one week after injection. The method can be applied within minutes to efficiently deliver transgenes into subcutaneous adipose tissues. Thus, this protocol allows for studying genes of interest in a timely manner in murine brown/beige fat and could potentially lead to new gene therapies for obesity. Brown adipose tissue is a special type of fat contributing to energy expenditure in human newborns and adults. Moreover, subcutaneous white adipose tissue has a high capacity to adapt an energy-consuming, brown-like/beige phenotype. Here, we developed an easy to handle and fast to accomplish method to efficiently transfer genes into brown and beige fat pads in vivo. Lentiviral vectors are directly injected into the target fat pad of anaesthetized mice through a small incision using a modified, small needle connected to a microsyringe, which is well suited for infiltration of adipose tissues. Expression of the target gene can be detected in brown/beige fat one week after injection. The method can be applied within minutes to efficiently deliver transgenes into subcutaneous adipose tissues. Thus, this protocol allows for studying genes of interest in a timely manner in murine brown/beige fat and could potentially lead to new gene therapies for obesity
A practical approach to remote ischemic preconditioning and ischemic preconditioning against myocardial ischemia/reperfusion injury
No full textAlthough urgently needed in clinical practice, a cardioprotective therapeutic approach against myocardial ischemia/reperfusion injury remains to be established. Remote ischemic preconditioning (rIPC) and ischemic preconditioning (IPC) represent promising tools comprising three entities: the generation of a protective signal, the transfer of the signal to the target organ, and the response to the transferred signal resulting in cardioprotection. However, in light of recent scientific advances, many controversies arise regarding the efficacy of the underlying signaling. We here show methods for the generation of the signaling cascade by rIPC as well as IPC in a mouse model for in vivo myocardial ischemia/reperfusion injury using highly reproducible approaches. This is accomplished by taking advantage of easily applicable preconditioning strategies compatible with the clinical setting. We describe methods for using laser Doppler perfusion imaging to monitor the cessation and recovery of perfusion in real time. The effects of preconditioning on cardiac function can also be assessed using ultrasound or magnetic resonance imaging approaches. On a cellular level, we confirm how tissue injury can be monitored using histological assessment of infarct size in conjunction with immunohistochemistry to assess both aspects in a single specimen. Finally, we outline, how the rIPC-associated signaling can be transferred to the target cell via conservation of the signal in the humoral (blood) compartment. This compilation of experimental protocols including a conditioning regimen comparable to the clinical setting should proof useful to both beginners and experts in the field of myocardial infarction, supplying information for the detailed procedures as well as troubleshooting guides
Protocol for evaluation of neurotrophic strategies in Parkinson
No full textParkinson’s disease (PD) is a neurodegenerative disease that is characterised by motor and non-motor symptoms which result from the progressive degeneration of nigrostriatal ventral midbrain (VM) dopaminergic (DA) neurons, as well as peripheral sympathetic neurons. PD is incurable, with current therapeutic strategies providing symptomatic relief. Neurotrophic factor (NTF) therapy has the potential to protect degenerating neurons in PD. However, there has been limited success in PD clinical trials due to neurotrophic strategies that are invasive, inefficient in delivering sustained neurotrophic support, do not protect all degenerating neurons and may have a compromised mechanism of action in the PD brain. Therefore, while neurotrophic therapy remains a promising disease-modifying approach for PD, it is important to identify novel neurotrophic strategies that can protect all neurons affected by PD. To address this need, we report an integrated approach for pre-clinical evaluation of potential neurotrophic strategies, e.g., pharmacological agents (e.g., drugs/small molecules), signalling proteins (e.g., morphogens) and/or genetic (gene/mRNA) modifications, in cellular models of the neuronal populations that are affected by PD. Herein, we describe, in detail, an in vitro protocol that allows a step-wise evaluation of the efficacy, and mechanism(s) of action, of novel neurotrophic strategies in VM DA neurons and sympathetic neurons, following an initial evaluation in a human cell line model of these cells, SH-SY5Y cells. The protocol uses the induction of neurite growth as the primary measure of neurotrophic action. Indeed, the neuro-protection/-restoration of PD-affected axons is widely thought to be an appropriate target for effective therapeutic intervention in PD
Incorporation of β-actin loading control into zymography
No full textGelatin zymography and immunoblot are widely used gel electrophoresis techniques to study matrix metalloproteinases-2 and -9. Each method has its advantages and disadvantages. Zymography is exquisitely sensitive but offers no loading control to ensure equal sample loading. Immunoblot is 100–1000-fold less sensitive, but allows for the probing of a sample loading control such as β-actin to ensure accurate protein loading. In this report, we describe two simple protocols that combine gelatin zymography to study MMP-2 and -9 levels with an in-gel β-actin immunoblot loading control, thus combining sensitivity and accuracy in a single assay. The protocols incorporate the loading of molecular weight markers to demarcate MMP-2/-9 from the β-actin. The first protocol utilizes the overlay of a 10% zymography gel over a 5% Tris-Glycine separating gel from which the β-actin is transferred. The second protocol involves the direct transfer of the β-actin from a single 10% zymography gel