Journal of biological methods最新文献

{"title":"Multi-parametric flow cytometry staining procedure for analyzing tumor-infiltrating immune cells following oncolytic herpes simplex virus immunotherapy in intracranial glioblastoma","authors":"P. Bommareddy, D. Lowe, H. Kaufman, S. Rabkin, D. Saha","doi":"10.14440/jbm.2019.281","DOIUrl":"https://doi.org/10.14440/jbm.2019.281","url":null,"abstract":"Multi-color flow cytometry is a standard laboratory protocol, which is regularly used to analyze tumor-infiltrating immune cell subsets. Oncolytic herpes simplex virus has shown promise in treating various types of cancers, including deadly glioblastoma. Intracranial/intratumoral treatment with oncolytic herpes simplex virus expressing interleukin 12, i.e., immunovirotherapy results in induction of anti-tumor immune responses and tumor infiltration of a variety of immune cells. Multi-color flow cytometry is employed to characterize immune cells in the tumor microenvironment. Here, we describe a step-by-step 11-color flow cytometry protocol to stain tumor-infiltrating immune cells in glioblastoma following oncolytic herpes virotherapy. We also describe a method to identify HSV-1 glycoprotein-B-specific CD8+ T cells using fluorochrome-conjugated major histocompatibility complex multimers. The multimers carry major histocompatibility peptide complexes, which have the ability to interact and bind to T cell receptors present on the surface of T cells; allowing identification of T cells (e.g., CD8+) reactive to a desired antigen. This multimer staining can be used in conjunction with the multi-parametric flow cytometry staining. Brain tumor quadrants are harvested, minced, enzymatically digested, immune cells are isolated by positive selection, single cells are counted and blocked for Fc receptors, cells are incubated with dye and/or color-conjugated antibodies, and flow cytrometry is performed using a BD LSRII flow cytometer. The protocol described herein is also applicable to stain immune cells in other mouse and human tumors or in any desired tissues.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47540283","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Real time mitochondrial dimension measurements","authors":"Joseph M. Leichner, E. Konyukhov, David Kamoun, Y. Yaniv","doi":"10.14440/jbm.2019.262","DOIUrl":"https://doi.org/10.14440/jbm.2019.262","url":null,"abstract":"Mitochondrial volume is correlated with cell function and internal cell processes. Changes in mitochondrial volume were associated with advanced states of cardiac disease. Thus, measurements of mitochondrial dimension deformations are important to the understanding of cell function and its deterioration. Existing methods either allow measurements of the volume of isolated mitochondria, which are an inferior model to that of isolated cells, or they allow short time measurements that are toxic to the cells. Recent studies have discovered that mitochondrial deformation along a given cell axis can be measured by using the Fourier transformation on the variation in transmitted light intensity induced by the periodic lattice of myofilaments alternating with mitochondrial rows. However, this method was used only offline and in a line scan mode, making it impossible to measure both axes. We designed an open source program in LabVIEW to take advantage of the transmitted light diffraction technique and quantify mitochondrial two dimension (2D) deformation in cardiomyocytes, in situ in real time for long periods (more than several seconds). We validated the program on synthetic and on experimental images from rabbit and rat ventricular myocytes. The program can analyze offline and real time simultaneous 2D mitochondrial deformation dynamics as well as also sarcomere length dynamics. Moreover, the program can accurately analyze images acquired from different cameras. Quantification of mitochondrial 2D deformations is a powerful tool for exploring cell biophysics and bioenergetics mechanisms and will lay the foundation for a future clinical tool for quantifying mitochondrial volume changes associated with different cardiac diseases.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46171165","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A step-by-step beginner’s protocol for whole genome sequencing of human bacterial pathogens","authors":"S. Gautam, Rajendra Kc, K. W. Leong, Micheál Mac Aogáin, R. O’Toole","doi":"10.14440/jbm.2019.276","DOIUrl":"https://doi.org/10.14440/jbm.2019.276","url":null,"abstract":"Bacterial whole genome sequencing (WGS) is becoming a widely-used technique in research, clinical diagnostic, and public health laboratories. It enables high resolution characterization of bacterial pathogens in terms of properties that include antibiotic resistance, molecular epidemiology, and virulence. The introduction of next-generation sequencing instrumentation has made WGS attainable in terms of costs. However, the lack of a beginner’s protocol for WGS still represents a barrier to its adoption in some settings. Here, we present detailed step-by-step methods for obtaining WGS data from a range of different bacteria (Gram-positive, Gram-negative, and acid-fast) using the Illumina platform. Modifications have been performed with respect to DNA extraction and library normalization to maximize the output from the laboratory consumables invested. The protocol represents a simplified and reproducible method for producing high quality sequencing data. The key advantages of this protocol include: simplicity of the protocol for users with no prior genome sequencing experience and reproducibility of the protocol across a wide range of bacteria.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-03-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.14440/jbm.2019.276","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45093644","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Inflammatory monocyte response due to altered wall shear stress in an isolated femoral artery model","authors":"Aparna A. Kadam, R. Gersch, T. Rosengart, M. Frame","doi":"10.14440/jbm.2019.274","DOIUrl":"https://doi.org/10.14440/jbm.2019.274","url":null,"abstract":"Arteriogenesis (collateral formation) is accompanied by a pro-inflammatory state that may be related to the wall shear stress (WSS) within the neo-collateral vessels. Examining the pro-inflammatory component in situ or in vivo is complex. In an ex vivo mouse femoral artery perfusion model, we examined the effect of wall shear stress on pro-arteriogenic inflammatory markers and monocyte adhesion. In a femoral artery model with defined pulsatile flow, WSS was controlled (at physiological stress, 1.4×, and 2× physiological stress) during a 24 h perfusion before gene expression levels and monocyte adhesion were assessed. Significant upregulation of expression was found for the cytokine TNFα, adhesion molecule ICAM-1, growth factor TGFβ, and the transcription factor Egr-1 at varying levels of increased WSS compared to physiological control. Further, trends toward upregulation were found for FGF-2, the cytokine MCP-1 and adhesion molecules VCAM-1 and P-selectin with increased WSS. Finally, monocytes adhesion increased in response to increased WSS. We have developed a murine femoral artery model for studying changes in WSS ex vivo and show that the artery responds by upregulating inflammatory cytokines, adhesion molecules and growth factors consistent with previous in vivo findings.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48769972","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A minimally-invasive serial cerebrospinal fluid sampling model in conscious Göttingen minipigs","authors":"A. Bergadano, E. M. Amen, B. Jacobsen, S. Belli, Anthony Vandjour, Christelle Rapp, C. Senn","doi":"10.14440/jbm.2019.265","DOIUrl":"https://doi.org/10.14440/jbm.2019.265","url":null,"abstract":"Drug concentrations in cerebrospinal fluid (CSF) are typically used as a as a surrogate measure of their availability in the CNS, and CSF penetration in animal studies are used for assessment of CNS drug delivery in early preclinical drug development. The minipig is a valid alternative to dogs and non-human primates as non-rodent species in preclinical research, but this species presents anatomical peculiarities that make the serial collection of CSF technically challenging. A minimally-invasive serial cerebrospinal fluid collection model via catheterization of the subarachnoid space in conscious minipigs was developed allowing assessment of longitudinal drug pharmacokinetics in the central nervous system in preclinical research. Shortly, the subarachnoid space was accessed in the anesthetized minipig by puncture with a Tuohy needle; when CSF was flowing through the needle a catheter was advanced and thereafter tunneled and fixed on the back. The PK of peptide A administered subcutaneously was performed and CSF could be sampled in the conscious animals for up to 48 h. When compared to the plasma kinetic data, there was a clear difference in the elimination phase of Pept. A from CSF, with an apparent longer average terminal half-life in CSF. The 3Rs are addressed by reducing the number of animals needed for a pharmacokinetic profile in central nervous system and by improving the validity of the model avoiding biases due to anesthesia, blood contamination, and inter-individual variability.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-01-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47966106","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Small RNA-seq: The RNA 5'-end adapter ligation problem and how to circumvent it.","authors":"Lodoe Lama,&nbsp;Jose Cobo,&nbsp;Diego Buenaventura,&nbsp;Kevin Ryan","doi":"10.14440/jbm.2019.269","DOIUrl":"https://doi.org/10.14440/jbm.2019.269","url":null,"abstract":"<p><p>The preparation of small RNA cDNA sequencing libraries depends on the unbiased ligation of adapters to the RNA ends. Small RNA with 5' recessed ends are poor substrates for enzymatic adapter ligation, but this 5' adapter ligation problem can go undetected if the library preparation steps are not monitored. Here we illustrate the severity of the 5' RNA end ligation problem using several pre-miRNA-like hairpins that allow us to expand the definition of the problem to include 5' ends close to a hairpin stem, whether recessed or in a short extension. The ribosome profiling method can avoid a difficult 5' adapter ligation, but the enzyme typically used to circularize the cDNA has been reported to be biased, calling into question the benefit of this workaround. Using the TS2126 RNA ligase 1 (a.k.a. CircLigase) as the circularizing enzyme, we devised a bias test for the circularization of first strand cDNA. All possible dinucleotides were circle-ligated with similar efficiency. To re-linearize the first strand cDNA in the ribosome profiling approach, we introduce an improved method wherein a single ribonucleotide is placed between the sequencing primer binding sites in the reverse transcriptase primer, which later serves as the point of re-linearization by RNase A. We incorporate this step into the ribosomal profiling method and describe a complete improved library preparation method, Coligo-seq, for the sequencing of small RNA with secondary structure close to the 5' end. This method accepts a variety of 5' modified RNA, including 5' monophosphorylated RNA, as demonstrated by the construction of a HeLa cell microRNA cDNA library.</p>","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"6 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6507418/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37234164","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Multi-parametric flow cytometry staining procedure for analyzing tumor-infiltrating immune cells following oncolytic herpes simplex virus immunotherapy in intracranial glioblastoma.","authors":"Praveen K Bommareddy,&nbsp;Devin B Lowe,&nbsp;Howard L Kaufman,&nbsp;Samuel D Rabkin,&nbsp;Dipongkor Saha","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Multi-color flow cytometry is a standard laboratory protocol, which is regularly used to analyze tumor-infiltrating immune cell subsets. Oncolytic herpes simplex virus has shown promise in treating various types of cancers, including deadly glioblastoma. Intracranial/intratumoral treatment with oncolytic herpes simplex virus expressing interleukin 12, <i>i.e</i>., immunovirotherapy results in induction of anti-tumor immune responses and tumor infiltration of a variety of immune cells. Multi-color flow cytometry is employed to characterize immune cells in the tumor microenvironment. Here, we describe a step-by-step 11-color flow cytometry protocol to stain tumor-infiltrating immune cells in glioblastoma following oncolytic herpes virotherapy. We also describe a method to identify HSV-1 glycoprotein-B-specific CD8⁺ T cells using fluorochrome-conjugated major histocompatibility complex multimers. The multimers carry major histocompatibility peptide complexes, which have the ability to interact and bind to T cell receptors present on the surface of T cells; allowing identification of T cells (<i>e.g</i>., CD8⁺) reactive to a desired antigen. This multimer staining can be used in conjunction with the multi-parametric flow cytometry staining. Brain tumor quadrants are harvested, minced, enzymatically digested, immune cells are isolated by positive selection, single cells are counted and blocked for Fc receptors, cells are incubated with dye and/or color-conjugated antibodies, and flow cytrometry is performed using a BD LSRII flow cytometer. The protocol described herein is also applicable to stain immune cells in other mouse and human tumors or in any desired tissues.</p>","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"6 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/9b/27/jbm-6-2-e112.PMC6528664.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37007451","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A rapid, high-throughput method for determining chronological lifespan in budding yeast","authors":"Z. Belak, T. Harkness, C. Eskiw","doi":"10.14440/jbm.2018.272","DOIUrl":"https://doi.org/10.14440/jbm.2018.272","url":null,"abstract":"The budding yeast Saccharomyces cerevisiae is a major model system in the study of aging. Like metazoans, yeast lifespan is extended by caloric restriction and treatment with pharmacological agents which extend lifespan. A major workhorse of aging research in budding yeast is the chronological lifespan assay. Traditionally, chronological lifespan assays consist of taking regular samples of aging yeast cultures, plating out aliquots on agar, and counting the resulting colonies. This method, while highly reliable, is labor-intensive and expensive in terms of materials consumed. Here, we report a novel MTT-based method for assessing chronological lifespan in yeast. We show that this method is equal to the colony counting method in its rigorous and reliable measurement of lifespan extension in yeast as a result of caloric restriction, and is able to distinguish known long-lived and short-lived yeast strains. We have further developed this method into a high-throughput assay that allows rapid screening of potential anti-aging compounds as well as yeast strains with altered lifespan. Application of this method permits the rapid identification of anti-aging activities in yeast and may facilitate identification of materials with therapeutic potential for higher animals and, most importantly, humans.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-12-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43081710","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High resolution measurement of membrane receptor endocytosis.","authors":"Zhihui Zhang, David K Heidary, Christopher I Richards","doi":"10.14440/jbm.2018.266","DOIUrl":"10.14440/jbm.2018.266","url":null,"abstract":"<p><p>We present a new approach to quantify the half-life of membrane proteins on the cell surface, through tagging the protein with the photoconvertible fluorescent protein, Dendra2. Upon exposure to 405 nm light, Dendra2 is photoconverted from green to red emission. Total internal reflection fluorescence microscopy (TIRF) is applied to limit visualization of fluorescence to proteins located on the plasma membrane. Conversion of Dendra2 works as a pulse chase experiment through monitoring only the population of protein that has been photoconverted. As the protein is endocytosed the red emission decreases due to the protein leaving the TIRF field of view. This method is not impacted by the insertion of new protein into the plasma membrane as newly synthesized protein only exhibits green emission. We used this approach to determine the half-life of ENaC on the plasma membrane illustrating the high temporal resolution capability of this technique compared to current methods.</p>","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":" ","pages":"e105"},"PeriodicalIF":0.0,"publicationDate":"2018-12-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6706155/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43010679","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Oxidation of cysteine-rich proteins during gel electrophoresis","authors":"C. Achilli, A. Ciana, G. Minetti","doi":"10.14440/jbm.2018.275","DOIUrl":"https://doi.org/10.14440/jbm.2018.275","url":null,"abstract":"The first electrophoretic analysis of proteins was performed in 1937 by Arne Tiselius [1], who was awarded the Nobel Prize in Chemistry 1948 for this important contribution. This fundamental analytical technique, which had a decisive impact on research in such disciplines as biochemistry, microbiology, immunology and molecular biology, has been perfected over the years. In present days, the most common type of electrophoretic separation of proteins is based on their preventive denaturation by sodium dodecyl sulfate (SDS). This amphipathic detergent binds to the denatured proteins in a well calibrated ratio, abolishes their original intrinsic net charge and confers to them a negative charge of uniform density. Proteins so treated are separated by polyacrylamide gel electrophoresis in SDS (SDS-PAGE). In the gel, they all migrate towards the anode, with an electrophoretic mobility correlated with their mass. Discontinuous SDS-PAGE was then introduced, by Leonard Ornstein and Baruch J. Davis in 1964 [2,3], as a more efficient variant of SDSPAGE. It consists of a stacking gel polymerized on top of the resolving gel. The stacking gel, by its more acidic pH and lower polyacrylamide concentration, allows proteins to stack into a thin band before entering into the resolving gel, where they separate as better resolved bands. Concerning the visualization of proteins in gel, two main strategies have taken hold: the non-specific protein staining with dyes such as coomassie brilliant blue [4], or the specific detection of a given protein by Western blotting [5]. In addition, an SDS-PAGE variant, later to be named zymography, was introduced for in-gel visualization of proteins endowed with hydrolytic activity, especially proteases. This method is based on incorporation into the polyacrylamide gel of a specific substrate for the protease under investigation. After electrophoretic separation, the gel is incubated in a suitable buffer to ensure that the proteases possibly present in the original sample acquire again their enzymatic activity and digest the substrate in situ. The gel is then stained, for instance with coomassie blue, and the sites of proteolysis appear as white bands on a blue background [6]. SDS-PAGE, as a method for separating proteins according to their mass, has been further improved by Ulrich K. Laemmli in 1970 [7]. In the new protocol the protein samples are denatured with SDS in the presence of 2-mercaptoethanol, a reducing agent that cleaves any disulfide bond, whether native or artificially induced, between cysteine residues in proteins. The compound also prevents subsequent oxidation of cysteines and maintains them in the reduced state. One year later, Grant Fairbanks et al. further perfected the protocol for analysis of erythrocyte membrane proteins, by replacing 2-mercaptoethanol with dithiothreitol, a dimercaptan reducing agent more powerful than 2-mercaptoethanol itself [8]. It is common conviction that during electrophoretic separatio","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48448912","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}