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Past Issue in 2013
Volume: 3, Issue: 17
Cancer Biology
Isolation of Mouse Tumor-Infiltrating Leukocytes by Percoll Gradient Centrifugation
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 Percoll density gradient, because Percoll possesses a low viscosity, a low osmolarity and no toxicity to cells. Different leukocyte populations are isolated based on their density and collected at the interface between 40% and 80% discontinuous Percoll gradient.
Three-dimensional Invasion Assay
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 describe a 3D invasion assay for observing the morphology and comparing the invasive ability of cancer cells in artificial 3D environments (Yang et al., 2012). Collagen I gels are used to cover on the top of cancer cells attached on coverslip glass dish and medium containing FBS is added as a chemoattractant. After incubation for a suitable time, the cells are fixed and stained. The invasion index can be calculated and the morphology can be imaged with a laser confocal microscope.
Chemosensitivity Assay
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 96-well culture plates or 24-well plates, and then different concentrations of anti-cancer drugs are added into each well. After that, measure the cell density or cell activity to observe the effect of anti-cancer drugs to cancer cells or cancer stem cells. The chemoresistance of cells is stronger, the cell density or cell activity is higher. Here, we describe chemosensitivity assays for osteosarcoma cells and osteosarcoma stem-like cells.
Genomic 8-oxo-7,8-dihydro-2'-deoxyguanosine Quantification
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 repair (reviewed in Delaney et al., 2012). The following assay is used to quantify 8-oxo-dGuo levels in DNA as described in Sousa et al. (2013).
3H-Penciclovir (3H-PCV) Uptake Assay
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 PET-imaging of various biological functions in small animals, by combining with radiolabeled substrates such as 18F-FHBG and 124I-FIAU. 3H-Penciclovir (PCV) uptake assay is a method of choice used to determine the expression level of HSV1-TK in mammalian cells and tissues. HSV1-TK phosphorylate PCV and result in the formation of penciclovir monophosphate, and its subsequent phopsphorylation by cellular TK lead to the formation of penciclovir triphosphate, which is trapped selectively in cells express HSV-TK. 3H-Penciclovir enables the detection of penciclovir uptake of mammalian cells and tissues by radioactive procedures such as scintillation counting. Here we describe the protocol to carry out 3H-Penciclovir uptakes in mammalian cells.
Immunology
Mouse Macrophage Differentiation by Induction with Macrophage Colony-Stimulating Factor
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 a simple method to induce the differentiation of mouse bone marrow-derived myeloid precusor cells into macrophages in the presence of M-CSF.
C1q Binding to and Uptake of Apoptotic Lymphocytes by Human Monocyte-derived Macrophages
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 autologous lymphocytes as a source of apoptotic cells. The use of autologous cells as the apoptotic target rather than transformed cell lines avoids antigenic stimulation from “nonself” structures at the HLA level but also from “altered self” signals due to the transformation inherent in cell lines.
Microbiology
Colony Immunoblotting Assay for Detection of Bacterial Cell-surface or Extracellular Proteins
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. The assay can distinguish bacterial clones with different expression rates (high, medium and low) from colonies that do not express target protein. We used this assay for screening of Mat fimbriae-producing Escherichia coli mutants obtained by mini-Tn5 transposon mutagenesis and immunomagnetic separation (Lehti et al., 2013).
Neuroscience
Generation of Mouse Spinal Cord Injury
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.
Preparation of Pre- and Post-synaptic Density Fraction from Mouse Cortex
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 fraction, and PSD fraction. It helps to identify the location of synaptic protein and find the potential synaptic complex.
Targeted Occlusion of Individual Pial Vessels of Mouse Cortex
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., 2011), as well as to generate localized ischemic strokes following occlusion of single penetrating vessels (Nishimura et al., 2007; Drew et al., 2010; Shih et al., 2013). The intravascular clot is formed by irradiation of a target vessel with a focused green laser after injection of a circulating photosensitizing agent, Rose Bengal (Watson et al., 1985). We briefly describe modifications of custom-designed and commercial two-photon imaging systems required to introduce a green laser for photothrombosis. We further provide instructions on how to occlude a single penetrating arteriole within the somatosensory cortex of an anesthetized mouse.
Plant Science
Seed Germination and Viability Test in Tetrazolium (TZ) Assay
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 transfer reaction catalysed by the cellular dehydrogenases. TZ enters both living and dead cells but only living cells catalyse the formation of formazan which being non-diffusible stains the viable seeds red whereas the absence of respiration prevents formazan production making the dead seeds (aged tissue) remain unstained.The brief description of this protocol has been reported in Verma et al., 2013.
Protein Extraction, Acid Phosphatase Activity Assays, and Determination of Soluble Protein Concentration
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 tissue- and/or cellular compartment-specific isozymes that display marked differences in their physical and kinetic properties. Increases in intracellular (vacuolar) and secreted APase activities are useful biochemical markers of plant nutritional Pi deficiency. The protocols for protein extraction, APase activity determination and measurement of soluble protein concentration from plant tissues or cell suspension cultures are presented.
Heterologous Production and Anaerobic Purification of His- and StrepII-tagged Recombinant Proteins
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 ATCC 824 are used, which are grown either aerobic or anaerobic with glucose. Two standard chromatographic methods for purification were applied using His- and StrepII-tagged proteins (Figure 1). All procedures have been performed in an anaerobic tent to avoid the access of oxygen.
Quantification of Total and Soluble Inorganic Phosphate
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 is performed with tissues extracted under mild acid conditions (which preserves acid-labile phosphate ester bonds).
Pectin Methylesterase Activity Assay for Plant Material
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 wall modification, as it promotes the formation of Ca2+cross-links along the stretches of the demethylesterified galacturonic acid residues (Wolf et al., 2009; Müller et al., 2013), and thus influences the biophysical properties of plant cell walls. Here, we describe a protocol that can be used to estimate the activity of PMEs in a total soluble protein extract from plant or seed tissues. Soluble protein is extracted from the plant/seed materials, and a coupled enzyme assay is performed, according to a procedure modified from Grsic-Rausch and Rausch (2004). The methanol released from methylesterified pectins as a result of PME activity is oxidized to formaldehyde by alcohol oxidase. The formaldehyde is then used as an electron donor by formaldehyde dehydrogenase to reduce NAD+ to NADH. The formation of NADH from NAD+ is followed spectrophotometrically, and used to estimate the PME activity in the protein extract.
Pyruvate:ferredoxin Oxidoreductase (PFR1) Activity Assays Using Methyl Viologen as Artificial Electron Acceptor
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 vivo transferred to ferredoxin.In this assay we use methyl viologen as artificial electron acceptor which turns into dark violet (ε604 = 13.6 mM-1 cm-1) (Mayhew, 1978) in its reduced state (Figure 1).
RNA Isolation From Meloidogyne Spp. Galls
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 protein and sugar contents). The protocol allows obtaining an RNA yield of 5-15 μg total RNA from 250-300 hand dissected galls at 3 days post infection (dpi) (Figure 1). It was proved particularly for Arabidopsis and tomato.
Plant Endo-β-mannanase Activity Assay
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 endo-β–mannanase assay. A high-salt buffer without thiol reductants is required for effective extraction of the enzyme. The enzyme is able to digest water-insoluble AZCL galactomannan to release water soluble dyed fragments, which is detected through measurement of absorbance at 590 nm wavelength. Increase in absorbance at 590 nm is correlated directly with enzyme activity.