Journal of Biological Methods (JBM)
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163 research outputs found
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A modified rat model of hindlimb ischemia for augmentation and functional measurement of arteriogenesis
No full textArteriogenesis (collateral artery development) is an adaptive pathway critical for salvage of tissue in the setting of arterial occlusion. Rodent models of arteriogenesis typically involve an experimental occlusion (ligation) of a hindlimb artery and then rely on indirect measures such as laser Doppler perfusion imaging to assess blood flow recovery. Unfortunately, the more commonly utilized measures of distal tissue perfusion at rest are unable to account for hemodynamic and vasoactive variables and thus provide an incomplete assessment of collateral network capacity. We provide a detailed description of modifications to the commonly used model of femoral artery ligation. These serve to alter and then directly assess collateral network’s hemodynamic capacity. By incorporating an arteriovenous fistula distal to the arterial ligation, arterial growth is maximized. Hindlimb perfusion may be isolated to measure minimum resistance of flow around the arterial occlusion, which provides a direct measure of collateral network capacity. Our results reinforce that arteriogenesis is driven by hemodynamic variables, and it can be reliably augmented and measured in absolute terms. Using these modifications to a widely used model, functional arteriogenesis may be more directly studied
Method for selective quantification of immune and inflammatory cells in the cornea using flow cytometry
No full textThe cornea serves as a protective surface against the environment (i.e., allergens, pollutants, desiccation and microorganisms) and promotes vision, made possible by corneal transparency. This protocol describes corneal preparation for flow cytometry to assess cells localized in the cornea. Our model details the process, from determining how many corneas are needed in the experiment to corneal excision to digestion and staining of the cornea cells. The simplicity of the model allows for systematic analysis of different corneal mechanisms of immunity, inflammation, angiogenesis and wound healing. In corneal transplantation, residential immune and inflammatory cells are key to the mechanisms that underlie angiogenesis, opacity, and graft rejection. In addition, this model can also elucidate cellular mechanisms mediating corneal graft outcomes and wound healing. Lastly, this model can be used to analyze the efficacy of new medications such as instillation and subconjunctival injections and assess the potential of therapeutic molecules to enhance graft survival and wound healing in vivo
Isolation, cultivation and immunostaining of single myofibers: An improved approach to study the behavior of satellite cells
No full textSatellite cells are myogenic cells responsible for muscle growth shortly after birth and muscle repair/regeneration during adulthood. Therapies based on satellite cells hold promise for treating muscular dysfunctions. Studying satellite cells is technically challenging owing to their low abundance, small size and anatomical dispersed location between the basal lamina and the sarcolemma of myofibers. In this article, we present three improved protocol strategies for studying the properties of satellite cells of the mouse during the different stages of muscle regeneration: (1) immunostaining of freshly isolated single myofibers to facilitate the study of quiescent satellite cells, (2) cultivation of single myofibers on Matrigel®-coated dish to study the myogenesis programs initiated by satellite cell activation, and (3) cultivation of single myofibers in floating conditions to analyze activated satellite cells or the doubling time of satellite cells in myofibers. In brief, when compared to previously published protocols, this article presented an improved protocol that requires shorter experimental time and less laborious approach for higher yield of intact single myofibers for downstream analyses
Development and validation of an allele-specific PCR assay for genotyping a promoter and exonic single nucleotide polymorphisms of MGMT gene
No full textDNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) specifically remove the methyl/alkyl group from the O6-position of guanine and restore the guanine to its normal form without causing DNA strand breaks. Relationship between MGMT activity and resistance to alkylating therapeutic agents is well established. Non-availability of simple, cost-effective and efficient methods of genotyping may hinder investigations on genotype-phenotype associations. No simple genotyping procedures such as allele-discrimination Taqman Assays were available for two genetic variations in MGMT gene that had previously demonstrated to be affecting its function and expression.These two variants were included to genotype in a clinical study (Clinicaltrail.gov ID: NCT01257854). Hence, the present study is aimed at developing, validating a rapid and simple allele-specific PCR method that genotypes exonic variant rs2308321 (c.520A>G) and a promoter variant rs113813075 (c.-459C>A) with standard PCR instruments.Web-based allele-specific (AS) primer design application called web-based allele-specific primer was used to design primers. Genomic DNA of lymphoblastoid cell line obtained from the Coriell repository with known genotypes were used to standardize the genotyping procedure. The PCR products were analyzed by 3% Agarose gel electrophoresis and by DNA Screen Tape assay with the Agilent 4200 TapeStation. The allele-specific PCR assay described here is a suitable strategy for efficient and reliable genotyping for difficult variants. This method offers cost-effective strategy for genotyping in clinical cohort studies provided positive controls established by Sanger sequencing are available for the variant.
RNA isolation from Peyer
No full textSampling and immune surveillance within gut-associated lymphoid tissues (GALT) such as the intestinal Peyer’s patch (PP) occurs by an elegantly orchestrated effort that involves the epithelial barrier, B and T lymphocytes, and an extensive network of mononuclear phagocytes. Although we now understand more about the dynamics of antigen and microbial sampling within PPs, the gene expression changes that occur in individual cell subsets during sampling are not well characterized. This protocol describes the isolation of high-quality RNA from sorted PP, B and T-lymphocytes, and CD11c+ phagocytes for use with nCounter-nanostring technology. This method allows investigators to study gene expression changes within PPs in response to antigens, microbes, and oral vaccine delivery vehicles of interest that are sampled
A model for the detection of pancreatic ductal adenocarcinoma circulating tumor cells
No full textMetastatic disease is the leading cause of pancreatic ductal adenocarcinoma (PDAC) associated death. PDAC cells invade and enter the bloodstream early, before frank malignancy can be detected. Our objective was to develop an in vivo assay enabling the identification and quantification of circulating tumor cells (CTCs) from primary orthotopic PDAC tumors. Human PDAC cells expressing luciferase and green fluorescent protein were orthotopically injected into the pancreas of mice utilizing ultrasound guidance. Bioluminescent imaging was conducted to identify and track tumor growth. CTCs were then isolated and analyzed by flow cytometry to detect GFP-expressing cancer cells. Tumor growth as measured by bioluminescent imaging increased over time. The concentration of CTCs correlated with the strength of bioluminescent imaging signal. In addition, livers bearing macroscopic disease were harvested for further imaging under fluorescence stereomicroscopy and confocal microscopy, which confirmed the presence of metastases. This study represents an orthotopic animal model that reliably detects the presence of CTCs from PDAC. There is a positive correlation between the concentrations of CTCs with overall tumor burden
A rapid, high-throughput method for determining chronological lifespan in budding yeast
No full textThe 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
Agrobacterium-mediated transformation of Camelina sativa for production of transgenic plants
No full textCamelina sativa (C. sativa), an oilseed species rich in poly-unsaturated fatty acids, has gained great importance as an industrial oil platform crop in recent years. Despite the potential benefits of C. sativa for bioenergy applications, limited research has been conducted to improve its agronomic qualities. Hence, a simple and efficient technique for production of transgenic C. sativa plants is warranted. In the present study, shoot apical meristems of two C. sativa cultivars (Pl650159 and Pl650161) were transformed with Agrobacterium strain ‘EHA 105’ harboring the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase II (nptII) genes. After two days of co-cultivation in the dark, explants were transferred to selection medium. Transgenic shoots were identified on the basis of green fluorescence and kanamycin resistance. Shoots were then rooted and transferred to potting mix soil for acclimatization. This protocol describes an efficient method to generate transgenic C. sativa plants in as little as 4 weeks
Insulin receptor based lymphocyte trafficking in the progression of type 1 diabetes
No full textThe insulin receptor (IR) is a transmembrane receptor which recognizes and binds the hormone insulin. We describe two models that were devised to explore the role of IR over-expression on T-lymphocytes and their chemotactic motility in the progression of type 1 diabetes. FVB/NJ-CD3-3×FLAG-mIR/MFM mice were generated to selectively over-express 3×FLAG tagged murine IR in T-lymphocytes via an engineered CD3 enhancer and promoter construct. Insertion of the 3×FLAG-mIR transgene into FVB/NJ mice, a known non-autoimmune prone strain, lead to a minor population of detectable 3×FLAG-mIR tagged T-lymphocytes in peripheral blood and the presence of a few lymphocytes in the pancreas of the Tg+/- compared to age matched Tg-/- control mice. In order to induce stronger murine IR over-expression then what was observed with the CD3 enhancer promoter construct, a second system utilizing the strong CAG viral promoter was generated. This system induces cell specific IR over-expression upon Cre-Lox recombination to afford functional 3×FLAG tagged murine IR with an internal eGFP reporter. The pPNTlox2-3×FLAG-mIR plasmid was constructed and validated in HEK-Cre-RFP cells to ensure selective Cre recombinase based 3×FLAG-mIR expression, receptor ligand affinity towards insulin, and functional initiation of signal transduction upon insulin stimulation
Rapid and affordable size-selected PacBio single-molecule real-time sequencing template library construction using the bead-beating DNA extraction method
No full textThis study demonstrated that bead-beating method facilitates a simple and rapid protocol for genomic DNA isolation for Pacific BioSciences (PacBio) sequencing with library construction of sufficient length. The protocol may also be beneficial for inactivating pathogens by simultaneous and instant DNA fragmentation, with no special equipment required to obtain large DNA fragments. This protocol was comparable in terms of quality to the standard protocol suggested by PacBioand represents an alternative, rapid shortcut for performing accurate PacBio sequencing