In vitro chemotaxis

Protein extraction To isolate biologically active protein, organs can be placed in 1 mL cold 50 mM Tris-HCl, pH 7.5, 0.2 mM EDTA and homogenized on ice. Extracts are clarified by centrifugation at 15,000 for 30 min, followed by a second centrifugation at 100,000 for 45 min. This results in a preparation of protein which, in the case of chemokines, can be used for in vitro chemotaxis assays to determine bioactivity. The presence of transgenic protein in these preparations can also be documented by immunoblot. 

Bridges RB, Kraal JH, Huang L JT, et al. 1977. Effects of cigarette smoke components on in vitro chemotaxis of human polymorphonuclear leukocytes. Infec Immun 16 240-248. 

The concept of leukocyte-specific chemoattractants was enunciated almost as soon as microscopy revealed the existence of leukocyte-specific inflammatory infiltrates (I). With the advent of reproducible in vitro chemotaxis assays, evidence for the existence of chemoattractants was bolstered by activities found in crude biological preparations. For example, the monocytic infiltrates associated with a wide variety of tumor types were suggested to be elicited by a tumor-derived chemotactic factor (TDCF) that could attract monocytes to the exclusion of neutrophils (2). In the late 1970s and early 1980s, a monocyte-specific chemoattractant activity could be demonstrated in medium conditioned by malignant cell lines whose cognate tumors were associated with monocytic infiltration in vivo (2-4). 

An activation of the complement cascade results in generation of the chemotactic cleavage product C5a. Through this activation, polysaccharides are able to increase leukocyte migration in an in vitro chemotaxis assay when incubated in the presence of serum [67, 68]. 

Suriyasathapom W., A.J.J.M. Daemen, E.N. Noordhuizen-Stassen, S.J. Dieleman, M. Nielen and Y.H. Schukken, 1999. p-hydroxybutyrate levels in peripheral blood and ketone bodies supplemented in culture media affect the in vitro chemotaxis of bovine leukocytes. Vet. Immunol. Immunopathol. 68, 177-186. 

The gold-standard assay used for all chemokine receptor inhibitors that reach clinical-phase trials is the chemotaxis functional assay. This assay relies on the ability of chemokines to recruit cells expressing their respective receptor to areas of inflammation. In vitro, this assay was first described in detail by Taub et al. (16) for 24/48-well plates currently, this can be achieved by using 96-well plates. Cells are incubated in the upper chamber with an antagonist for a particular receptor (at different concentrations or with buffer) and challenged to migrate to the lower chamber, which has the relevant chemokine. After 2 to 4 hours of incubation at 37°C, the upper chamber inlet is removed and the cells in the lower chamber quantified by fluorescence with, for example, Calcein AM (Invitrogen, Carlsbad, CA). 

Several opiate receptors have been identified on cells of the nervous systems of animals and humans, with mu (p), kappa (k), and gamma (y) subtypes being predominant. These classical opiate receptors are G- protein coupled 7-transmembrane molecules.27 Opiates predominantly affect immune responses directly by ligation of p, k, and y opiate receptors, as well as non-classical opiate-like receptors, on immune cells and indirectly by binding to receptors on CNS cells. Studies conducted in vitro with opiate-treated immune cells demonstrated receptor-mediated reduced phagocytosis, chemotaxis and cytokine and chemokine production. These effects are linked to modulation of host resistance to bacterial, protozoan, viral and fungal infections using animal models, cell lines and primary cells. 

The formylated peptide fMet-Leu-Phe is probably the most commonly-used activator of neutrophils in vitro. It is used as a model agonist to study receptor-mediated processes, generating intracellular signalling molecules that then activate cell functions. This compound can, depending upon the concentration used, activate many varied functions, such as chemotaxis, aggregation, reactive oxidant production, cytoskeletal changes and (particularly in combination with cytochalasin B) degranulation. 

The uPA receptor also appears to play a role in cell locomotion and chemo-taxis, at least for human monocytes in vitro (G8). Using these cells, chemotaxis was inhibited by pretreatment with both anti-uPA receptor antibodies and anti-sense uPA receptor oligonucleotides. Expression of uPA catalytic activity was not required for chemotaxis with this cell type (G8). 

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