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Cancer Biology

Genomic 8-oxo-7,8-dihydro-2'-deoxyguanosine Quantification Author:  Antonio Sarno, date: 09/05/2013, view: 3935, Q&A: 0
8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo) is among the most common reactive oxygen species-induced DNA lesions and can be used as a biomarker for oxidative stress. The lesion has been linked to several biological processes and diseases, including colorectal cancer, Huntington’s disease, estrogen-induced gene expression, and thymine dimer ...
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Three-dimensional Invasion Assay Authors:  Wen-Hao Yang and Muh-Hwa Yang, date: 09/05/2013, view: 4580, Q&A: 0
The invasive ability of cancer cells is a crucial function for cancer metastasis and the surrounding microenvironment of cancer cells in living tissues is three-dimension (3D). Therefore, to establish an in vitro invasion assay in a 3D system to predict cancer invasive ability is valuable in the research for cancer metastasis. Here, we ...
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3H-Penciclovir (3H-PCV) Uptake Assay Authors:  Thillai V Sekar and Ramasamy Paulmurugan, date: 09/05/2013, view: 3657, Q&A: 0
Thymidine Kinase from human Herpes simplex virus type 1 (HSV1-TK) in combination with specific substrate prodrug nucleotide analogue ganciclovir (GCV) has been widely used as suicidal therapeutic gene for cancer gene therapy. HSV1, and its mutant (HSV1-sr39TK) with improved substrate specificity, were used as reporter genes for ...
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Chemosensitivity Assay Authors:  Haixia Zhang and Yan Zhang, date: 09/05/2013, view: 4915, Q&A: 0
Chemoresistance is one of the special properties of cancer stem cells, which is the main cause of chemotherapy failure and plays an important role in the recurrence of various cancers including osteosarcoma. The most widely used assay for evaluating chemoresistance is the modified cell proliferation assay. An equal number of cells are seeded onto ...
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Isolation of Mouse Tumor-Infiltrating Leukocytes by Percoll Gradient Centrifugation Authors:  Ying Liu, Keqiang Chen, Chunyan Wang, Wanghua Gong, Teizo Yoshimura, Ji Ming Wang and Mingyong Liu, date: 09/05/2013, view: 17168, Q&A: 0
A hallmark of cancer-associated inflammation is the infiltration of leukocytes into tumors, which is believed to be recruited by chemokines. Some infiltrating leukocytes such as macrophages often promote tumor growth by producing growth-inducing and angiogenic factors. Here, we described a method of isolating tumor-infiltrating leukocytes with ...
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Immunology

C1q Binding to and Uptake of Apoptotic Lymphocytes by Human Monocyte-derived Macrophages Authors:  Marie E. Benoit, Elizabeth V. Clarke and Andrea J. Tenner, date: 09/05/2013, view: 4019, Q&A: 0
To characterize macrophage gene expression profiles during the uptake of autologous apoptotic cells, we developed a unique, more physiologic system using primary human monocyte derived macrophages purified via a nonactivating isolation procedure (and in the absence of contaminating platelets, which can release stimulating signals if activated) and ...
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Mouse Macrophage Differentiation by Induction with Macrophage Colony-Stimulating Factor Authors:  Ying Liu, Keqiang Chen, Chunyan Wang, Wanghua Gong, Teizo Yoshimura, Ji Ming Wang and Mingyong Liu, date: 09/05/2013, view: 4725, Q&A: 0
Macrophages are differentiated from circulating blood monocytes and act as tissue-resident professional phagocytes. Macrophages function in both innate and adaptive immune systems of vertebrate animals. The cytokine macrophage colony-stimulating factor (M-CSF) is essential for the proliferation and differentiation of monocytes. Here, we described ...
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Microbiology

Colony Immunoblotting Assay for Detection of Bacterial Cell-surface or Extracellular Proteins Authors:  Timo A. Lehti and Benita Westerlund-Wikström, date: 09/05/2013, view: 11475, Q&A: 1
This simple protocol describes how to detect antigens from agar-grown bacterial colonies transferred to nitrocellulose using specific antibodies. The protocol is well suitable for detection of bacterial proteins exposed on the cell surface or secreted to the extracellular space and it can be modified also for detection of intracellular proteins. ...
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Neuroscience

Preparation of Pre- and Post-synaptic Density Fraction from Mouse Cortex Authors:  Chengyong Shen and Yong-Jun Chen, date: 09/05/2013, view: 5748, Q&A: 0
The understanding of the organization of postsynaptic signaling systems at excitatory synapses has been aided by the identification of proteins in the postsynaptic density (PSD) fraction, a subcellular fraction enriched in structures with the morphology of PSDs. Here we described an efficient way to isolate the crude synaptosome, presynaptic ...
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Generation of Mouse Spinal Cord Injury Authors:  Oneil G. Bhalala, Liuliu Pan, Hilary North, Tammy McGuire and John A. Kessler, date: 09/05/2013, view: 9039, Q&A: 1
Spinal cord injury (SCI) is a debilitating injury with significant morbidity and mortalitiy. Understanding the pathogensis of and developing treatments for SCI requires robust animal models. Here we describe a method for generating an efficient and reproducible contusion model of SCI in adult mice.
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Targeted Occlusion of Individual Pial Vessels of Mouse Cortex Authors:  Zachary J. Taylor and Andy Y. Shih, date: 09/05/2013, view: 4501, Q&A: 0
Targeted photothrombosis is a method to occlude individual arterioles and venules that lie on the surface of the cerebral cortex. It has been used to study collateral flow patterns within the pial vascular network following occlusion of single surface vessels (Schaffer et al., 2006; Blinder et al., 2010; Nguyen et al., ...
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Plant Science

RNA Isolation From Meloidogyne Spp. Galls Authors:  Alejandra García, Marta Barcala, Javier Cabrera, Carmen Fenoll and Carolina Escobar, date: 09/05/2013, view: 5101, Q&A: 0
We describe an efficient method to obtain a sufficient quantity of RNA from nematode-induced galls with a high quality and integrity, proved to be appropriate for transcriptomic analysis, i.e. real time PCR, microarray hybridization or second generation sequencing. This protocol is efficient for small quantities of galls (organs with high ...
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Heterologous Production and Anaerobic Purification of His- and StrepII-tagged Recombinant Proteins Author:  Jens Noth, date: 09/05/2013, view: 7535, Q&A: 0
This protocol describes the heterologous expression and purification of proteins related to anoxic hydrogen production of Chlamydomonas reinhardtii (Noth et al., 2013). For this, the bacterial expression hosts Escherichia coli BL21 (DE3) ΔiscR (Akhtar MK et al., 2008) and Clostridium acetobutylicum ...
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Pyruvate:ferredoxin Oxidoreductase (PFR1) Activity Assays Using Methyl Viologen as Artificial Electron Acceptor Author:  Jens Noth, date: 09/05/2013, view: 5546, Q&A: 0
Here we describe the activity measurements of heterologous expressed pyruvate:ferredoxin oxidoreductase (Noth et al., 2013) (Noth et al.,2013) from Chlamydomonas reinhardtii. This enzyme catalyzes the reversible reaction (I) from pyruvate to acetyl CoA and CO2 generating low potential electrons which are in ...
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Plant Endo-β-mannanase Activity Assay Authors:  Yunjun Zhao and Laigeng Li, date: 09/05/2013, view: 4326, Q&A: 0
Endo-β–mannanases in plant require post-translational modification, such as N-glycosylation and disulfide-linked dimerization, for their catalytic activity. Determination of the plant endo-β–mannanase activity needs to modify the assay conditions for optimizing their enzymatic reaction. Here, we describe a modified method for plant ...
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Seed Germination and Viability Test in Tetrazolium (TZ) Assay Authors:  Pooja Verma and Manoj Majee, date: 09/05/2013, view: 20962, Q&A: 3
Tetrazolium (TZ) assay is the fast evaluation for seed viability and alternative quick method for seed’s germinability (Porter et al., 1947; Wharton, 1955). All respiring tissues are capable of converting a colourless compound, TZ (2,3,5 triphenyl tetrazolium chloride) to a carmine red coloured water-insoluble formazan by hydrogen ...
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Protein Extraction, Acid Phosphatase Activity Assays, and Determination of Soluble Protein Concentration Authors:  Vicki Knowles and William Plaxton, date: 09/05/2013, view: 8819, Q&A: 0
Acid phosphatases (APases) catalyze the hydrolysis of inorganic phosphate (Pi) from a broad range of Pi-monoesters with an acidic pH optimum. The liberated Pi is reassimilated into cellular metabolism via mitochondrial or chloroplastic ATP synthases of respiration or photosynthesis, respectively. Eukaryotic APases exist as a wide variety of ...
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Quantification of Total and Soluble Inorganic Phosphate Authors:  Vicki Knowles and William Plaxton, date: 09/05/2013, view: 5905, Q&A: 0
A simple, rapid, and sensitive colorimetric microassay for inorganic phosphate (Pi) relies upon the absorption at 660 nm of a molybdenum blue complex that forms upon reduction of an ammonium molybdate-Pi complex in acid. The method for determination of total Pi uses plant tissues that have been ashed at 500 °C, whereas quantification of soluble Pi ...
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Pectin Methylesterase Activity Assay for Plant Material Authors:  Kerstin Mueller, Sebastian Bartels and Allison R. Kermode, date: 09/05/2013, view: 4827, Q&A: 0
Homogalacturonans, the most abundant pectins of the plant cell wall, can be methylesterified at the C-6 position of the galacturonic acid residues. Demethylesterification of cell wall pectins is catalyzed by apoplastic pectin methylesterases (PMEs). Several plant developmental processes and plant-environment interactions involve PME-mediated cell ...
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