Cell Migration in Inflammation and Immunity by Daniele D'Ambrosio, Francesco Sinigaglia

By Daniele D'Ambrosio, Francesco Sinigaglia

Chemokines and their receptors play a significant function within the pathogenesis of diverse acute and persistent inflammatory illnesses. This booklet offers useful protocols for the examine of chemokine receptor biology.

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J. Immunol. 152, 5495–5502. 28. , et al. (2000) Molecular characterization of dipeptidyl peptidase activity in serum: soluble CD26/dipeptidyl peptidase IV is responsible for the release of X-Pro dipeptides. Eur. J. Biochem. 367, 5608–5613. 29. , et al. (2001) Kinetic investigation of chemokine truncation by CD26/dipeptidyl peptidase IV reveals a striking selectivity within the chemokine family. J. Biol. Chem. 276, 29,839–29,845. 30. , et al. (1999) Structure–activity relationship of diaryl phosphonate esters as potent irreversible dipeptidyl peptidase IV inhibitors.

4; 50 mM NaCl and loaded on a heparin–Sepharose column. 05–2 M NaCl) and the protein concentration was measured by a Coomassie blue G-250 protein assay, using bovine serum albumin as a standard (A). Fractions were tested at various concentrations for neutrophilic granulocyte chemotactic activity (expressed in units/mL) in the migration test under agarose (26). One unit corresponds to the amount of granulocyte chemotactic protein (GCP) that results in a half-maximal migration distance under agarose.

47 Add 600 µL of the chemokine solution (see Note 3) to the wells of the 24-well plate (see Note 4). To have duplicates, two sets of three wells each are set up for every chemokine dilution. Twelve wells should be designated as negative controls and receive 600 µL of migration medium only. These wells are used to determine the background or “spontaneous” migration (see Note 5). Dispense 100 µL of CD4+ T-cells into the transwell insert and quickly but gently transfer them to their original position onto the 24-well plate.

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