Adhesion Protein Protocols by Amanda S. Coutts

By Amanda S. Coutts

The moment variation of Adhesion Protein Protocols combines conventional recommendations with state-of-the-art and novel recommendations that may be tailored simply to assorted molecules and telephone varieties. the themes mentioned during this up-to-date moment version comprise novel ideas for learning cell-cell adhesion, neutrophil chemotaxis, in vitro assays used to review leukocyte migration via monolayers of cultured endothelial cells, and novel strategies to purify pseudopodia from migratory cells.

This publication additionally discusses the learn of cell-matrix interplay, RNA interference, fluorescence restoration after photobleaching, actin purification, the applying of microarray thoughts, and the function of adhesion proteins within the research of proteomics.

The protocols mentioned during this quantity are appropriate for either amateur and professional scientists, who will achieve extra perception into the advanced and incompletely understood methods keen on mobile adhesion.

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2. Growth and Maintenance of HL-60 Cells To generate stable cell lines and facilitate proper differentiation, HL-60 cells must be carefully maintained and passaged within a midlog range of growth. This requires the cells to be passaged approximately every 2 d. 1. Growth and Passage 1. Using a hemocytometer, count the HL-60 cells. 2. Resuspend the HL-60 cells at 2 × 105 cells/mL in 20–30 mL HL-60 growth medium and place them in a T75 tissue culture flask. 3. Cells are maintained at 37°C and 5% CO2.

The transwell assay can be used to examine random cell motility or chemokinesis with soluble attractant in both the upper and lower compartments of the Transwell chamber or, alternatively, to examine chemotaxis or directed migration toward higher concentrations of soluble attractant in the lower compartment of the chamber only. The functional readout of the assay is the number of cells that migrate to the bottom chamber in a specific time period and does not provide information about the migration phenotype that may contribute to altered chemotaxis.

J. (2001) Sec6/8 complexes on trans-Golgi network and plasma membrane regulate late stages of exocytosis in mammalian cells. J. Cell Biol. 55, 593–604. 9. Vogelmann, R. and Nelson, W. J. (2005) Fractionation of the epithelial apical junctional complex: reassessment of protein distributions in different substructures. Mol. Biol. Cell 16, 701–716. Analysis of Neutrophil Chemotaxis 23 3 Analysis of Neutrophil Chemotaxis Paul A. Nuzzi, Mary A. Lokuta, and Anna Huttenlocher Summary Neutrophils are the initial responders to bacterial infection or other inflammatory stimuli and comprise a key component of the innate immune response.

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