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Past Issue in 2015
Volume: 5, Issue: 2
Immunology
Phenotyping of Live Human PBMC using CyTOFTM Mass Cytometry
Single-cell analysis has become an method of importance in immunology. Fluorescence flow cytometry has been a major player. However, due to issues such as autofluorescence and emission spillover between different fluorophores, alternative techniques are being developed. In recent years, mass cytometry has emerged, wherein antibodies labeled with metal ions are detected by ICP-MS. In order for a cell to be seen, a metal in the mass window must be present; there is no analogous parameter to forward or side scatter. The current mass window selected is approximately AW 103-196, which includes the lanthanides used for most antibody labeling, as well as iridium and rhodium for DNA intercalators.In this protocol, we use a cocktail of antibodies labeled with MAXPAR metal-chelating polymers to surface-stain live PBMC that have been previously cryopreserved. Many of these markers were taken from a standard fluorescence phenotyping panel (Maecker et al., 2012). No intracellular antibodies are used. We use a CyTOFTM (Cytometry by Time-Of-Flight) mass cytometer to acquire the ICP-MS data. Subsequent analysis of the dual count signal data using FlowJo software allows for cell types to be analyzed based on the dual count signal in each mass channel. The percentage of each cell type is determined and reported as a percent of the parent cell type.
Isolation of CNS-infiltrating and Resident Microglial Cells
Variety of immunological and biochemical studies associated with infection or inflammation in the central nervous system (CNS) utilize CNS-resident and/or infiltrating cells which were isolated from the CNS of naïve and affected mice in order to investigate the underlying mechanisms and the potential roles of the cell populations. Mechanical and enzyme-based single cell preparations of CNS cells are subjected to a density gradient to obtain functional single cells. In combination with cell-specific biomarkers, the function and/or status of resident microglia and infiltrating lymphocytes, including B and T cells as well as macrophages, can be characterized.
Microbiology
Determination of the Secondary Structure of an RNA fragment in Solution: Selective 2`-Hydroxyl Acylation Analyzed by Primer Extension Assay (SHAPE)
This protocol describes the methodology for the determination of the secondary structure of an RNA fragment in solution using Selective 2´-Hydroxyl Acylation analyzed by Primer Extension, abbreviation SHAPE. It consists in the very fast chemical modification of flexible and therefore possibly single-stranded nucleotides in a sequence-independent manner using benzoyl cyanide (BzCN), forming 2´-O-adducts. The modifications in the RNA are then analyzed by primer extension. Reverse transcriptase is blocked by the 2´-O-adducts formed. The advantage of the method is, first, that not each RNA molecule studied but the primer used in the extension reaction is labelled and, second, that the resulting cDNA analyzed in sequencing gels is much more stable than the modified RNA.
In vitro Dynamic Model of a Catheterized Bladder and Biofilm Assay
Biofilm formation on catheters is thought to contribute to persistence of catheter-associated urinary tract infections (CAUTI) which represent the most frequent nosocomial infections. Understanding of factors relevant for CAUTI pathogenesis and evaluation of new therapeutics or interference strategies requires a model system that mirrors the physico-chemical conditions prevailing in a catheterized human bladder. The described in vitro dynamic model of a catheterized bladder enables to emulate many of the characteristics of a catheterized human bladder albeit in the absence of a bladder epithelium. A minor modification compared to the original model system (Stickler, et al., 1999) allows temperature maintenance of the top 10 cm of the catheter, thereby enabling reproducible monitoring of biofilm formation on the internal catheter surface.
Loading of Cells with Fluorescent Probe to Study Intracellular Acid-base Homeostasis in Lactic Acid Bacteria
Here we describe a protocol which we have used to study the homeostasis intracellular in vivo in lactic acid bacteria (LAB) using a fluorescent probe. This type of probes can be used for determining changes in the pH of cytoplasm with high sensitivity, temporal resolution and technical simplicity as well as accessing the rate of change of intracellular pH in response to a stimulus from kinetic measurements on short time scales (Breeuwer et al., 1996; Molenaar et al., 1991). This protocol has been designed to measure the intracellular pH using the pH-sensitive fluorescent probe 2´,7´-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) in LAB, Enterococcus faecalis (E. faecalis), Lactococcus lactis (L. lactis) and Lactobacillus casei (L. casei).
Mitochondrial Biogenesis Assay after 5-day Treatment in PC-3 Cells
Drug-induced mitochondrial injury can be caused by many different mechanisms including inhibition of mitochondrial DNA replication, transcription, translation, and altered protein function. Determination of the level of mitochondrial protein synthesis, or mitochondrial biogenesis, relative to the cellular protein synthesis, provides important information on potential mitochondrial toxicity.
Molecular Biology
Determination of Mitochondrial DNA Upon Drug Treatment
Drug-induced mitochondrial injury can be caused by many different mechanisms including inhibition of mitochondrial DNA replication, transcription, translation, and altered protein function. Determination of the level of mitochondrial DNA relative to the nuclear DNA levels provides important information on potential mitochondrial toxicity.
Plant Science
Camalexin Quantification in Arabidopsis thaliana Leaves Infected with Botrytis cinerea
Phytoalexins are heterogeneous low molecular mass secondary metabolites with antimicrobial activity produced at the infection site in response to pathogen invasion and represent an important part of the plant defense repertoire. Camalexin (3-Thiazol-2′-yl-indole) is a known phytoalexin first detected and isolated in Camelina sativa, from which it takes its name, infected with Alternaria brassicae (Browne et al., 1991). Production of camalexin is also induced in Arabidopsis thaliana leaves by a range of biotrophic and necrotrophic plant pathogens (bacteria, oomycetes, fungi and viruses) (Ahuja et al., 2012) as well as by abiotic stresses, such as UV and chemicals (e.g. acifluorfen, paraquat, chlorsulfuron and α-amino butyric acid) (Zhao et al., 1998; Tierens et al., 2002). Camalexin originates from tryptophan and CYP79B2 and CYP71B15 (PAD3) are P450 enzymes that catalyze important steps in its biosynthetic pathway (Glawischnig, 2007). The detection and quantification of camalexin content is required to understand how it is produced upon various stress conditions. Here we describe an easy method for camalexin extraction from Arabidopsis leaves infected with the necrotrophic fungus Botrytis cinerea, and further determination of camalexin levels by liquid chromatography–mass spectrometry (LC-MS). The method is sensitive enough to trace amount of camalexin down to the low pico-gram (10 pg/mg FW) range.
Stem Cell
PBMC-MSC Co-cultures for Induction of Treg Generation
To assess the capacity of multipotent stromal cells (MSC) to induce the generation of Tregs, transwell co-cultures were performed as well as cultures with MSC-conditioned medium (CM). In short, peripheral blood mononuclear cells (PBMC) were co-cultured with allogeneic MSC or CM for one week followed by one week of culture in the absence of MSC.
Monocyte-MSC Co-cultures
To assess the effect of multipotent stromal cells (MSC) on monocytes, 3-day cultures were performed of freshly isolated monocytes in MSC-conditioned medium (CM). As a control condition, monocytes were stimulated with low dose macrophage colony-stimulating factor (M-CSF). Monocytes were isolated from peripheral blood mononuclear cell (PBMC) populations by magnetic activated cell sorting (MACS) using CD14 microbeads.