Isolation of human and porcine monocytes and lymphocytes by three parameter flow sorting

Monocytes and lymphocytes were separated from total human blood cells by Ficoll-paque gradient centrifugation (at 600 g) followed by two parameter fluorescence activated cell sorting (forward [FLS] and perpendicular light scatter [PLS]). For human blood cells this technique gives good separation and high purity of the monocytes (more than 85%). For porcine blood cells we modified the Ficoll-paque gradient centrifugation-step by centrifuging at a lower speed (250 g). Since two parameter flow cytometry of porcine leukocytes (FLS and PLS) gave poor resolution we added endogenous non-specific esterase activity as a third parameter using fluorescein diacetate (FDA) as a fluorogenic substrate. The sorted fractions were cytocentrifuged and purity was checked with hematoxylin and/or peroxidase staining. Moreover, monoclonal antibodies against monocyte cell surface antigens were used to evaluate the purity of the sorted fractions. Three parameter sorting (PLS, FLS and fluorescence) yields good purification of porcine monocytes (86 +/- 1% pure) and lymphocytes (81 +/- 2% pure). There was a substantial adhesion of porcine monocytes (326 +/- 25/mm2) and lymphocytes (146 +/- 21/mm2) to monolayers of porcine aorta endothelial cells (PAEC).