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Past Issue in 2013
Volume: 3, Issue: 10
Biochemistry
Insulin Tolerance Test and Hyperinsulinemic-euglycemic Clamp
The two tests are used to evaluate in vivo sensitivity to insulin in mouse. The hypoerinsulinemic-euglycemic clamp provides information about the sensitivity to insulin in liver and other metabolically relevant tissues.
Stem Microsome Preparation and Mannan Synthase Activity Assay
Mannans are hemicellulosic polysaccharides and are present in cell walls of all land plants. Mannan polysaccharides are synthesized by two enzymes, mannan synthase (ManS) for backbone (mannan or glucomannan) synthesis and galactomannan galactosyl transferase for side-chain (galactosyl) addition. Here, a method for ManS activity assay using microsomes freshly isolated from Arabidopsis stems is described. This method can be applied to isolation of microsomes from any tissues of Arabidopsis or any other plants.
Immunology
Isolation of Infiltrating Leukocytes from the Spinal Cord of Mice
The infiltration of leukocytes into the central nervous system (CNS) is a common feature of many neuroinflammatory diseases such as multiple sclerosis, and also occurs during certain microbial infections such as by West Nile Virus. Here, we describe a method to isolate leukocytes from the spinal cords of mice. This method can be used for the characterization of leukocyte populations that infiltrate the spinal cord, and to perform functional studies with the isolated cells. The CNS of naive mice is infiltrated by very low numbers of leukocytes, however, upon inflammation increased numbers of mononuclear cells traffic to the CNS. The number of leukocytes that can be isolated roughly correlates with the degree of inflammation.
Systems Biology
DNA Methylation Profiling Using Infinium Methylation Assay
The Infinium Human Methylation 450 BeadChip technology allows the rapid quantitative DNA methylation analysis of more than 485,000 CpG dinucleotides located across the genome. The method utilizes sodium bisulfite treatment of genomic DNA to convert unmethylated cytosine residues into uracils whereas methylated cytosines remain unchanged. Modified DNA is then whole genome amplified, fragmented and hybridized to locus-specific oligomer probes linked to individual beads on a BeadChip. Hybridization is followed by single-base extension of the oligomer with a labeled nucleotide. The BeadChip is subsequently fluorescently stained and scanned to measure the intensities of the beads corresponding to the unmethylated and methylated CpG sites.