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Past Issue in 2013
Volume: 3, Issue: 8
Cell Biology
Flow Cytometric Detection of Reactive Oxygen Species
Reactive oxygen species (ROS) are molecules containing hydroxyl radicals or peroxides with unpaired electrons. In healthy aerobic cells, ROS are produced naturally as a byproduct of oxidative phosphorylation, oxidoreductase enzymes, or metal catalyzed oxidation at a controlled rate. However, ROS can be induced under some stress conditions especially exposure to environmental oxidants and certain drugs that leads to oxidative stress. Exceed ROS can cause damages in the building blocks of cells including DNA, proteins, and lipids, and eventually results in cell death. Cell-permeant 2', 7'-dichlorodihydrofluorescein diacetate (H2DCFDA) is a widely used ROS indicator. The reduced non-fluorescent fluorescein H2DCFDA can be oxidized and converted into fluorescent 2’, 7’-dichlorofluorescein (DCF) by intracellular ROS. In this protocol, we applied H2DCFDA to label the intracellular ROS and detected the DCF intensity by flow cytometry.
Flow Cytometric Detection of Mitochondrial Membrane Potential
Mitochondrial membrane potential (Δψm) is an important parameter of mitochondrial function and an indicator of cell health. Depletion of Δψm suggests the loss of mitochondrial membrane integrity reflecting the initiation of the proapoptotic signal. Recently, lipophilic cationic fluorescent dyes have been developed to detect Δψm by accumulating in the mitochondrial matrix until the Nernstian equilibrium distribution of lipophilic cations is reached. In this protocol, we applied a cell-permeant, green-fluorescent, lipophilic dye 3,3'-dihexyloxacarbocyanine Iodide (DiOC6(3)) which accumulates in mitochondria due to their large negative membrane potential, it can be applied to monitor the mitochondrial membrane potential using flow cytometric detection.
Immunology
Evaluation of Caspase-1 Activation and IL-1β Production in A Kainic Acid Microdyalisis Brain Injury Model
Intracerebral infusion of kainic acid (KA) by a microdialysis probe induces a focal swelling in the brain-perfused area which promotes inflammation (Compan et al., 2012; Oprica et al., 2003). The microdialysis technique allows the local in vivo perfusion of KA and the simultaneous collection of inflammatory mediators, and other neuroactive substances, released in the injured brain. This protocol also allows the perfusion of different solutions in each cerebral hemisphere at the same time. By perfusing KA in isotonic solution of Krebs-Ringer Bicarbonate (KRB) (280-290 mOsm) in one hippocampus and KA in hypertonic KRB solution (1,400-1,500 mOsm) in the contralateral side, we can evaluate in vivo the efficiency of hypertonic solutions in preventing inflammation induced by swelling after KA infusion. Once the inflammatory response has been induced, it is possible to infuse through the microdialysis probe a biotinylated specific inhibitor of caspase-1 allowing the detection of the brain regions and cells involved in IL-1 production in response to the injury (Oprica et al., 2003).
Microbiology
Packaging of Retroviral RNA into Viral Particles Analyzed by Quantitative Reverse Transcriptase-PCR
Formation of viral particles and packaging of genomic retroviral RNA into these particles are important steps in the late phase of the viral replication cycle. The efficiency of the incorporation of viral or cellular RNAs into viral particles can be studied using a quantitative Reverse Transcriptase-PCR (RT-qPCR)-based approach. After isolation of cytoplasmic RNA from either infected or transfected cells and extraction of virus particle-associated RNA, specific RNA levels present in both fractions are determined. The ratio of virion-associated and cytoplasmic RNA defines the encapsidation efficiency (Brandt et al., 2007; Blissenbach et al., 2010; Grewe et al., 2012).
Plasmodium falciparum Rosette Formation Assay
Rosetting, i.e. the capacity of red blood cells (iRBCs) infected with mature parasite stages to bind two or more uninfected red blood cells (RBCs) is a virulence factor of Plasmodium falciparum. This protocol describes an in vitro assay to monitor rosette formation by P. falciparum-infected red blood cells, including procedures for rosette enrichment, maintenance of rosetting phenotype and assays for rosetting with RBC labeled using lipophilic fluorescent probes.
Plasmodium falciparum Rosette Disruption Assay
Rosetting, i.e. the capacity of Plasmodium falciparum-infected red blood cells (iRBCs) to bind two or more uninfected red blood cells (RBCs) is associated with severe malaria in African children. Disruption of rosettes using small soluble inhibitors or specific antibodies is viewed as an interesting strategy to treat or prevent severe malaria manifestations. The protocol presented here describes an assay to monitor rosette dissociation, validated for the Palo Alto VarO, IT4/R29 and 3D7/PF13 rosetting clones (Vigan-Womas et al., 2011).
Molecular Biology
Detection and Cloning of Spliced Transcripts by RT-PCR
Using a Reverse Transcriptase-PCR approach spliced transcripts can be converted to cDNA, amplified and cloned into an expression plasmid. Sequencing of the obtained cDNA allows identification of the splicing events that generated the detected RNA (Grewe et al., 2012).
High-throughput Method for Determination of Seed Paternity by Microsatellite Markers
In this protocol, determination of seed paternity by microsatellite markers in Nicotiana attenuata is described. However, this does not include a protocol for the novel marker selection/identification, but rather exploits the markers generated for a closely related species N. tabacum (Bindler et al., 2007). This is a high-throughput protocol optimized and streamlined for one skilled person to process 384 (96 x 4) seeds in 5 days, from DNA isolation (from seedlings) to paternity assessment by microsatellite genotype data.
p65 Chromatin Immunoprecipitation Protocol
Chromatin Immunoprecipitation (ChIP) is an important procedure that allows you to verify if a certain protein is physically located at a regulatory region. This information, taken together with other procedures such as luciferase assays and EMSAs, will give definitive proof that the query protein is involved in the transcription of a protein. This procedure for p65 ChIP can be adapted to investigate other proteins; just a change of the antibody will suffice. The transcription factor known as NF-κB is a homo- or hetero-dimer consisting of members of the Rel/NFKB family. The most abundant NF-κB complexes are made of two different proteins, p65 (Rel-A) and p50 (NFKB1). The NF-κB complex is initially inhibited by IκB by direct binding, thus trapping NF-κB in the cytoplasm. After a stimulatory signal, IκB kinase (IKK) phosphorylates IκB, allowing IκB to undergo proteasome-mediated degradation. The degradation of IκB and phosphorylation of p65 by multiple kinases activates NF-κB, allowing it to transport to the nucleus and cause the transcriptional activation of many of its target genes containing κB sites (consensus sequence: gggRNNYYcc, R = purine Y = pyrimidine), such as PUMA, IL-6, and TNF.
Neuroscience
Primary Culture of Cortical Neurons
Primary culture of neurons from cerebral cortex is a popular model to study neuronal function in vitro and to explore the molecular mechanism of neurite outgrowth in the developing and adult central nervous system. This protocol is for preparing a culture of cerebral cortical neurons from postnatal rodent brain (Muramatsu et al., 2012). One day after cell plating, we can observe neurite outgrowth by microscope.
Culture of Rat Olfactory Ensheathing Cells Using EasySep® Magnetic Nanoparticle Separation
Olfactory ensheathing cells (OECs) can be isolated and purified from a range of postnatal day 7-day to 10-day rat olfactory bulbs. Rat OECs express the CD271/p75NTR receptor and using the “Do-It-Yourself” magnetic nanoparticle EasySep kit from STEMCELL technologies this protocol allows the selective purification of these cells in less than 50 min. Similar procedure can be used for mouse cultures.
Plant Science
Determination of Nectar Nicotine Concentration in N. attenuata
In this protocol, the determination of the nicotine concentration in nectar of Nicotiana attenuata is described. This method is applicable for the investigation of small amounts of nectar (above 1 μl). It is a high-throughput protocol optimized and streamlined for one skilled person to process approximately 100 nectar samples per day.