A rapid method for monocyte isolation and chemotaxis

Abstract

A rapid method for isolating human peripheral blood monocytes and studying chemotaxis is described. The cells are isolated at a mean purity of 60% (+/- 10%) using Dextran. T500 sedimentation of whole blood, followed by centrifugation of the leucocyte-rich plasma obtained on a discontinuous Percoll gradient (density 1.067 g/ml). Chemotaxis was performed using a 48-well chamber and the chemoattractants activated serum (C5a) and FMLP and was proven by checkerboard analysis. Using this technique pooled serum was found to be activated by the PVP-coated polycarbonate filter in use, producing the attractant C5a. At high concentrations this produced a chemotactic response identical to zymosan-activated serum (ZAS). This technique requires small volumes of blood (15 ml) and enables monocyte isolation and chemotaxis to be completed in approximately 4 hr using leucocyte preparations in which the monocyte is the predominant cell. It therefore facilitates the sequential investigation of monocyte function in patients.