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Generation of Giant Unilamellar Vesicles (GUVs) Using Polyacrylamide Gels

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Flow-cytometric Detection of Low-level Reactive Oxygen Species in Cell Lines and Primary Immune Cells

Authors: Kevin Bode, Corinna Link, Peter H. Krammer and Heiko Weyd, date: 09/05/2020, view: 2220, Q&A: 0

[Abstract] Depending on its concentration and cellular origin the production of reactive oxygen species (ROS) in the organism serves a variety of functions. While high concentrations during an oxidative burst are used to fight pathogens, low to moderate amounts of ROS act as signaling molecules important for several physiological processes such as regulation ...

Detection of Intracellular Reduced (Catalytically Active) SHP-1 and Analyses of Catalytically Inactive SHP-1 after Oxidation by Pervanadate or H₂O₂

Authors: Seeyoung Choi and Paul E. Love, date: 01/05/2018, view: 4238, Q&A: 0

[Abstract] Oxidative inactivation of cysteine-dependent Protein Tyrosine Phosphatases (PTPs) by cellular reactive oxygen species (ROS) plays a critical role in regulating signal transduction in multiple cell types. The phosphatase activity of most PTPs depends upon a ‘signature’ cysteine residue within the catalytic domain that is maintained in the ...

Measurement of Dipeptidylpeptidase Activity in vitro and in vivo

Authors: Rosa Barreira da Silva, Molly A. Ingersoll and Matthew L. Albert, date: 03/20/2017, view: 6196, Q&A: 0

[Abstract] Dipeptidylpeptidases (DPPs) are serine proteases, which cleave small proteins and peptides possessing a proline or an alanine in the second position of their N-terminus. Among the members of this family, dipeptidylpeptidase 4 (DPP4) is constitutively expressed in the extracellular space. DPP4 is found at the surface of many hematopoietic and ...

In vitro Treatment of Mouse and Human Cells with Endogenous Ligands for Activation of the Aryl Hydrocarbon Receptor

Authors: Taisho Yamada and Akinori Takaoka, date: 01/05/2017, view: 5781, Q&A: 0

[Abstract] Activation of the aryl hydrocarbon receptor (AHR) by endogenous ligands has been implicated in a variety of physiological processes such as cell cycle regulation, cell differentiation and immune responses. It is reported that tryptophan metabolites, such as kynurenine (Kyn) and 6-formylindolo(3,2-b)carbazole (FICZ), are endogenous ligands for AHR ...

Determination of H₂O₂ Generation by pHPA Assay

Authors: Jennifer L. Larson-Casey and A. Brent Carter, date: 11/20/2016, view: 6253, Q&A: 0

[Abstract] The production of reactive oxygen species, including H₂O₂, is a process that can be used in signaling, cell death, or immune response. To quantify oxidative stress in cells, a fluorescence technique has been modified from a previously described method to measure H₂O₂ release from cells (Panus et al ...

Reconstruction of the Mouse Inflammasome System in HEK293T Cells

Authors: Hexin Shi, Anne Murray and Bruce Beutler, date: 11/05/2016, view: 7561, Q&A: 0

[Abstract] The NLRP3 (NLR family, Pyrin domain containing 3) inflammasome is a multiprotein complex comprised of NLRP3, pro-caspase-1, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and the protein kinase NIMA related kinase 7 (NEK7) (Shi et al., 2016; He et al., 2016; Schmid-Burgk et al., ...

Quantification of Tumor Material Uptake

Authors: Richard N. Hanna and Catherine C. Hedrick, date: 10/20/2016, view: 6614, Q&A: 0

[Abstract] Extracellular tumor material including exosomes, microvesicles and apoptotic tumor debris may help cancers invade new organs. Enhancing the removal of extracellular tumor material by immune cells represents a novel immunotherapy approach for preventing cancer metastasis. This protocol quantifies the uptake and removal of extracellular tumor ...

In vivo Imaging of Tumor and Immune Cell Interactions in the Lung

Authors: Richard N. Hanna, Grzegorz Chodaczek and Catherine C. Hedrick, date: 10/20/2016, view: 6789, Q&A: 0

[Abstract] Immunotherapy has demonstrated great therapeutic potential by activating the immune system to fight cancer. However, little is known about the specific dynamics of interactions that occur between tumor and immune cells. In this protocol we describe a novel method to visualize the interaction of tumor and immune cells in the lung of live mice, ...

Lipid Extraction from HeLa Cells, Quantification of Lipids, Formation of Large Unilamellar Vesicles (LUVs) by Extrusion and in vitro Protein-lipid Binding Assays, Analysis of the Incubation Product by Transmission Electron Microscopy (TEM) and by Flotation across a Discontinuous Sucrose Gradient

Authors: Amina Bittame, Jodie Lopez, Gregory Effantin, Nicolas Blanchard, Marie-France Cesbron-Delauw, Jean Gagnon and Corinne Mercier, date: 10/20/2016, view: 11788, Q&A: 0

[Abstract] Dissecting the interactions established between proteins and membranes in a given type of cells is not an easy task. Using a cell-free system of large unilamellar vesicles (LUVs) to analyze these interactions may help decipher these interactions and identify potential membrane deformations induced by the proteins incubated with these LUVs. This ...

Measurement of mRNA Decay in Mouse Embryonic Fibroblasts

Authors: Lian-Qun Qiu, Wi S. Lai, Deborah J. Stumpo and Perry J. Blackshear, date: 07/05/2016, view: 9173, Q&A: 0

[Abstract] mRNA stability control is a critical step in the post-transcriptional regulation of gene expression. Actinomycin D, an antibiotic initially used as an anti-cancer drug, has turned out to be a convenient tool for studying the turnover rates of transcripts in cells, due to its inhibition of mRNA synthesis. Here, we describe a protocol for the ...

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