de Gruijter M, van Rijn M A, Verkerk A, Jongkind J F
Department of Cell Biology and Genetics, Erasmus University Rotterdam.
Zentralbl Veterinarmed A. 1990 Sep;37(8):585-91. doi: 10.1111/j.1439-0442.1990.tb00948.x.
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).
通过Ficoll-泛影葡胺梯度离心法（600 g），随后进行双参数荧光激活细胞分选（前向[FLS]和垂直光散射[PLS]），从人全血细胞中分离出单核细胞和淋巴细胞。对于人血细胞，该技术能实现良好的分离效果，且单核细胞纯度较高（超过85%）。对于猪血细胞，我们通过以较低速度（250 g）离心来改进Ficoll-泛影葡胺梯度离心步骤。由于猪白细胞的双参数流式细胞术（FLS和PLS）分辨率较差，我们添加了内源性非特异性酯酶活性作为第三个参数，使用荧光素二乙酸酯（FDA）作为荧光底物。将分选后的组分进行细胞离心涂片，并用苏木精和/或过氧化物酶染色检查纯度。此外，使用针对单核细胞表面抗原的单克隆抗体来评估分选组分的纯度。三参数分选（PLS、FLS和荧光）可实现猪单核细胞（纯度为86±1%）和淋巴细胞（纯度为81±2%）的良好纯化。猪单核细胞（326±25/mm²）和淋巴细胞（146±21/mm²）与猪主动脉内皮细胞（PAEC）单层有大量黏附。
